Preventing Post-Ictal Headaches

ABSTRACT

Disclosed herein are methods of preventing incidence of a migrainous or non-migrainous post-ictal headache, and/or at least one secondary symptom associated with a migrainous post-ictal headache in a subject comprising administering to the subject a monoclonal antibody that modulates the CGRP pathway. Compositions for use in the disclosed methods are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. Application No. 62/466,158, filed on Mar. 2, 2017; U.S. Application No. 62/490,953, filed Apr. 27, 2017; U.S. Application No. 62/514,346, filed Jun. 2, 2017; and U.S. Application No. 62/516,406, filed Jun. 7, 2017. The content of each of the foregoing applications is hereby incorporated by reference in its entirety.

BACKGROUND

Seizures affect about 50 million people worldwide and can be followed with headaches in up to about 50% of subjects. Ictal headaches are headaches associated with seizure activity. They may occur either before (pre-ictal) or after (post-ictal) a seizure, and in rare circumstances during a seizure. Many cases of ictal headache are misdiagnosed as a migraine with aura, or even a cluster headache. However, while those conditions usually involve only one side of the head (are unilateral), an ictal headache can be centrally situated or cover the entirety of the head. Post-ictal headaches (PIH) can be migrainous or non-migrainous in nature. A migrainous PIH is a PIH that is followed by a headache that fulfill migraine criteria, while a non-migrainous PIH is followed by a headache that does not fulfill migraine criteria.

Whether migrainous or non-migrainous in nature, PIHs add an additional significant burden to persons with epilepsy in addition to actual seizures (Schon et al., J. Neurol. Neurosurg. Psychiatry 50:1148-52, 1987; Toldo et al., J. Headache Pain 11:235-40, 2010; Wawrzyniak et al., Epilepsia 50 (Suppl 6):37, 2009). PIHs are very painful, and since they strike repetitively, PIHs cause subjects to endure significant hardship. Nonetheless, PIHs are mentioned only briefly in the latest epilepsy reference literature (see, e.g., Engel et al., Epilepsy: a comprehensive textbook. 2nd ed. Philadelphia: Wolters Kluwer/Lippincott Williams & Wilkins, 2008; and Wyllie E. The treatment of epilepsy: principles and practice. 3rd ed. Philadelphia: Lippincott Williams & Wilkins, 2001), and approved therapies for PIH are nonexistent. Of note, anti-epilepsy drugs, e.g., carbamazepine, clonazepam, lacosamide, nitrazepam, oxcarbazepine, phenobarbital, retigabine, tiagabine, and topiramate, have been reported to be successful in reducing migraine frequency, though they are unsuccessful for preventing PIHs. Accordingly, compositions and methods for preventing PIH are desired.

SUMMARY

Disclosed herein are anti-CGRP antibodies (e.g., anti-CGRP antagonist antibodies and anti-CGRP receptor antibodies) and methods of using the same for preventing a PIH, e.g., migrainous PIH and non-migrainous PIH. Also disclosed herein are methods of preventing, treating, or reducing incidence of PIH in a subject, e.g., a subject who is at risk for a PIH, comprising administering to the subject a therapeutically effective amount of a monoclonal antibody that modulates the CGRP pathway.

Methods of preventing, treating, or reducing incidence of at least one secondary symptom associated with migrainous PIH in a subject comprising administering to the subject a therapeutically effective amount of a monoclonal antibody that modulates the CGRP pathway are also provided.

In some embodiments of any of the methods described herein, the subject who is at risk for a post-ictal headache has a brain infection, traumatic brain injury, personal history of a seizure, family history of epilepsy, stroke, or dementia. In some embodiments, the subject is a human.

In some embodiments, the monoclonal antibody is an anti-CGRP antagonist antibody. In some embodiments, the monoclonal antibody is an anti-CGRP receptor antibody. The monoclonal antibody can be a human or humanized antibody (e.g., a humanized anti-CGRP antagonist antibody or a humanized anti-CGRP receptor antibody).

In some embodiments, the monoclonal antibody is administered intravenously. In some embodiments, the monoclonal antibody is administered subcutaneously.

In some embodiments, the monoclonal antibody is an IgG1, an IgG2, an IgG3, or an IgG4 antibody.

In some embodiments, the monoclonal antibody is administered to the subject from or using a pre-filled syringe, a pre-filled syringe with a needle safety device, an injection device, or an autoinjector comprising a dose of the monoclonal antibody.

In some embodiments, the methods described herein further comprise administering one or more additional doses of the monoclonal antibody to the subject.

In some embodiments, the monoclonal antibody comprises a CDR H1 as set forth in SEQ ID NO:3; a CDR H2 as set forth in SEQ ID NO:4; a CDR H3 as set forth in SEQ ID NO:5; a CDR L1 as set forth in SEQ ID NO:6; a CDR L2 as set forth in SEQ ID NO:7; and a CDR L3 as set forth in SEQ ID NO:8. In some embodiments, the monoclonal antibody comprises a heavy chain variable region comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:1, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the monoclonal antibody comprises a heavy chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:11, and a light chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:12. In some embodiments, the monoclonal antibody is fremanezumab. In some embodiments, the monoclonal antibody is administered at a dose of from about 225 mg to about 900 mg (e.g., at a dose of about 225 mg, at a dose of about 675 mg, at a dose of about 900 mg). These doses may be administered to the patient monthly or quarterly. Further, any of these doses (e.g., about 225, about 675, or about 900 mg) may be administered intravenously or subcutaneously. In a particular embodiment, the dosing regimen comprises an initial dose (e.g., 675 mg), and further comprises administering to the patient an additional dose of about 225 mg of the monoclonal antibody once per month in each of the two months (or three months, four months, five months, six months, or twelve months) subsequent to the month in which the initial dose is administered to the patient.

In some embodiments, the monoclonal antibody is administered as a formulation (e.g., a liquid formulation) comprising the antibody at a concentration of at least about 150 mg/mL. In another embodiment, the monoclonal antibody is administered in a volume of less than 2 mL (e.g., about 1.8 mL, about 1.7 mL, about 1.6 mL, about 1.5 mL, about 1.4 ml, about 1.3 mL, about 1.2 mL, about 1.1 mL, about 1.0 ml, about 0.9 mL, about 0.8 mL, about 0.7 mL, about 0.6 mL, about 0.5 mL, or less). In some embodiments, the monoclonal antibody is preferably administered in a volume of about 1.5 mL.

In an embodiment, the monoclonal antibody comprises a CDR H1 as set forth in SEQ ID NO:87; a CDR H2 as set forth in SEQ ID NO:88; a CDR H3 as set forth in SEQ ID NO: 89; a CDR L1 as set forth in SEQ ID NO:84; a CDR L2 as set forth in SEQ ID NO:85; and a CDR L3 as set forth in SEQ ID NO:86. In some embodiments, the monoclonal antibody comprises a heavy chain variable region comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:82, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:80. In some embodiments, the monoclonal antibody comprises a heavy chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:83, and a light chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:81.

In a further embodiment, the monoclonal antibody is eptinezumab. Eptinezumab may be administered at a dose of about 100 mg, about 300 mg, or about 1000 mg. Any of these doses (e.g., about 100 mg, about 300 mg, or about 1000 mg) may be administered intravenously or subcutaneously.

In another embodiment, the monoclonal antibody comprises a CDR H1 as set forth in SEQ ID NO:93; a CDR H2 as set forth in SEQ ID NO:94; a CDR H3 as set forth in SEQ ID NO:95; a CDR L1 as set forth in SEQ ID NO:91; a CDR L2 as set forth in SEQ ID NO:92; and a CDR L3 as set forth in SEQ ID NO:90. In some embodiments, the monoclonal antibody comprises a heavy chain variable region comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:97, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:96. In some embodiments, the monoclonal antibody comprises a heavy chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:99, and a light chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:98.

In a further embodiment, the monoclonal antibody is galcanezumab. Galcanezumab may be administered at a dose of about 120 mg or about 240 mg. Further, the 120 mg dose may be administered in a volume of about 1.5 mL and the 240 mg dose may be administered in a volume of about 3 mL. Any of these doses (e.g., about 120 mg or 240 mg) may be administered intravenously or subcutaneously.

In another embodiment, the monoclonal antibody comprises a CDR H1 as set forth in SEQ ID NO:103; a CDR H2 as set forth in SEQ ID NO:104; a CDR H3 as set forth in SEQ ID NO:105; a CDR L1 as set forth in SEQ ID NO:100; a CDR L2 as set forth in SEQ ID NO:101; and a CDR L3 as set forth in SEQ ID NO:102. In some embodiments, the monoclonal antibody comprises a heavy chain variable region comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:107, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:106. In some embodiments, the monoclonal antibody comprises a heavy chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:109, and a light chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:108.

In a further embodiment, the monoclonal antibody is erenumab. Erenumab may be administered at a dose of about 70 mg or about 140 mg. Further, the 70 mg does may be administered in a volume of about 1 mL. The 140 mg dose may be administered in a volume of about 2 mL. Any of these doses (e.g., about 70 or 140 mg) may be administered intravenously or subcutaneously.

In some embodiments of any of the methods described herein, the monoclonal antibody is administered prior to a seizure. For example, in some embodiments, the monoclonal antibody is administered at least about four hours (e.g., about five, six, seven, eight, nine, ten, eleven, twelve, twenty-four, thirty-six, forty-eight, seventy-two hours, or more) prior to a seizure.

In some embodiments, the monoclonal antibody, e.g., an anti-CGRP antagonist antibody, may be administered prior to a seizure, or can be before, during, and/or after any event likely to give rise to a seizure, e.g., at least about five minutes prior to a seizure, e.g., at least about ten minutes, at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about one hour, at least about two hours, at least about three hours, at least about four hours, at least about five hours, at least about six hours, at least about seven hours, at least about eight hours, at least about nine hours, at least about ten hours, at least about 12 hours, at least about 18 hours, at least about 24 hours, at least about 48 hours, or at least about 72 hours prior to a seizure. In some instances, the anti-CGRP antagonist antibody may be administered about two hours to about six hours prior to a seizure, e.g., about 2.5 hours to about 5.5 hours, about three hours to about five hours, or about 3.5 hours to about 4.5 hours prior to a seizure. For example, the anti-CGRP antagonist antibody may be administered about four hours prior to a seizure. In some instances, the anti-CGRP antagonist antibody may be administered about two, three, five, or six hours prior to a seizure.

In some embodiments, the methods described herein further comprises administering to the subject a second agent simultaneously or sequentially with the monoclonal antibody. The second agent can be non-steroidal anti-inflammatory drugs (NSAIDs) and/or triptans and/or a 5 hydroxytryptamine 1F receptor agonist (i.e., a serotonin receptor agonist). In some embodiments, the second agent is an agent taken by the subject prophylactically. In some embodiments, monthly use of the second agent by the subject is decreased by at least about 15%, e.g., at least 16%, 17%, 18%, 20%, 22%, 25%, 28%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least about 95%, after administering the monoclonal antibody.

Non-limiting examples of NSAIDs that can be used in combination with an anti-CGRP antibody include aspirin, diclofenac, diflusinal, etodolac, fenbufen, fenoprofen, flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac, tolmetin or zomepirac, cyclooxygenase-2 (COX-2) inhibitors, celecoxib, rofecoxib, meloxicam, JTE-522, L-745,337, NS398, or a pharmaceutically acceptable salt thereof.

Non-limiting examples of triptans that can be used in combination with an anti-CGRP antibody include sumatriptan, zolmitriptan, naratriptan, rizatriptan, eletriptan, almotriptan, and afrovatriptan.

A non-limiting example of a 5 hydroxytryptamine 1F receptor agonist is lasmiditan.

In another aspect, provided herein is the use of a monoclonal antibody that modulates the calcitonin gene related peptide (CGRP) pathway, for the manufacture of a medicament for prevention of a post-ictal headache.

In some embodiments, the monoclonal antibody comprises a CDR H1 as set forth in SEQ ID NO:3; a CDR H2 as set forth in SEQ ID NO:4; a CDR H3 as set forth in SEQ ID NO:5; a CDR L1 as set forth in SEQ ID NO:6; a CDR L2 as set forth in SEQ ID NO:7; and a CDR L3 as set forth in SEQ ID NO:8. In some embodiments, the monoclonal antibody comprises a heavy chain variable region comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:1, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the monoclonal antibody comprises a heavy chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:11, and a light chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:12.

In some embodiments, the monoclonal antibody comprises a CDR H1 as set forth in SEQ ID NO:87; a CDR H2 as set forth in SEQ ID NO:88; a CDR H3 as set forth in SEQ ID NO:89; a CDR L1 as set forth in SEQ ID NO:84; a CDR L2 as set forth in SEQ ID NO:85; and a CDR L3 as set forth in SEQ ID NO:86. In some embodiments, the monoclonal antibody comprises a heavy chain variable region comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:82, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:80. In some embodiments, the monoclonal antibody comprises a heavy chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:83, and a light chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:81.

In some embodiments, the monoclonal antibody comprises a CDR H1 as set forth in SEQ ID NO:93; a CDR H2 as set forth in SEQ ID NO:94; a CDR H3 as set forth in SEQ ID NO:95; a CDR L1 as set forth in SEQ ID NO:91; a CDR L2 as set forth in SEQ ID NO:92; and a CDR L3 as set forth in SEQ ID NO:90. In some embodiments, the monoclonal antibody comprises a heavy chain variable region comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:97, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:96. In some embodiments, the monoclonal antibody comprises a heavy chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:99, and a light chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:98.

In some embodiments, the monoclonal antibody comprises a CDR H1 as set forth in SEQ ID NO:103; a CDR H2 as set forth in SEQ ID NO:104; a CDR H3 as set forth in SEQ ID NO:105; a CDR L1 as set forth in SEQ ID NO:100; a CDR L2 as set forth in SEQ ID NO:101; and a CDR L3 as set forth in SEQ ID NO:102. In some embodiments, the monoclonal antibody comprises a heavy chain variable region comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:107, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:106. In some embodiments, the monoclonal antibody comprises a heavy chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:109, and a light chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:108.

Also disclosed are methods of decreasing a number of monthly headache or migraine hours experienced by a subject having PIH. In one embodiment, the method comprises administering to the subject an amount of a monoclonal antibody that modulates the CGRP pathway, wherein the monoclonal antibody is in an amount effective to decrease the number of monthly headache or migraine hours by at least 20 (e.g., 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or more headache or migraine hours) after a single dose. In some embodiments, the number of monthly headache or migraine hours is reduced by at least about 50 hours. In one embodiment, the method comprises administering to the subject an amount of a monoclonal antibody that modulates the CGRP pathway, wherein the monoclonal antibody is in an amount effective to decrease the number of monthly headache or migraine hours by at least 15% (e.g., 20%, 25%, 30%, 35%, 40%, or more) after a single dose. In some embodiments, the number of monthly headache or migraine hours is reduced by at least about 30%. In some embodiments, monthly headache or migraine hours experienced by the subject after said administering is reduced by 40 or more hours (e.g., 45, 50, 55, 60, 65, 70, 75, 80, or more) from a pre-administration level in the subject. Monthly headache or migraine hours may be reduced by more than 60 hours. In some embodiments, monthly headache or migraine hours experienced by the subject after said administering are reduced by 25% or more (e.g., 30%, 35%, 40%, 45%, 50%, or more) relative to a pre-administration level in the subject. Monthly headache or migraine hours may be reduced by 40% or more. In some embodiments, monthly headache or migraine days experienced by the subject after said administering is reduced by three or more days (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more days) from a pre-administration level in the subject. In some embodiments, the number of monthly headache or migraine days can be reduced by at least about 50% from a pre-administration level in the subject. Thus, in some aspects, the number of monthly headache or migraine days can be reduced by at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or at least about 90%.

Also disclosed are methods of decreasing a number of monthly headache or migraine days experienced by a subject having PIH. In one embodiment, the method comprises administering to the subject an amount of a monoclonal antibody that modulates the CGRP pathway, wherein the monoclonal antibody is in an amount effective to decrease the number of monthly headache or migraine days by at least 3 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more headache or migraine days) after a single dose. In some embodiments, the number of monthly headache or migraine days is reduced by at least about 6 headache or migraine days. In some embodiments, the number of monthly headache or migraine days can be reduced by at least about 50% from a pre-administration level in the subject. Thus, in some aspects, the number of monthly headache or migraine days can be reduced by at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or at least about 90%.

Also disclosed are methods of decreasing use of any acute headache medication in a subject having PIH, comprising administering to the subject a monoclonal antibody (e.g., an anti-CGRP antagonist antibody or an anti-CGRP receptor antibody) that modulates the CGRP pathway, wherein the monoclonal antibody is in an amount effective to decrease monthly use of the anti-headache medication by the subject by at least 15% (e.g., 20%, 25%, 30%, 35%, 40%, or more). In some embodiments, the anti-headache medication can be non-steroidal anti-inflammatory drugs (NSAIDs) and/or triptans. In some embodiments, the anti-headache medication is a triptan.

In one aspect, the invention provides a method of preventing, treating, or reducing incidence of PIH in a subject comprising administering to the subject a single dose of a monoclonal antibody (e.g., monoclonal anti-CGRP-antagonist antibody or an anti-CGRP receptor antibody) in an amount that modulates the CGRP pathway, wherein the amount of the monoclonal antibody is about 225 mg to about 1000 mg, e.g., about 675 mg or about 900 mg.

In a further aspect, the invention provides methods for preventing, treating, ameliorating, controlling, reducing severity of, reducing incidence of, or delaying the development or progression of PIH in an individual comprising administering to the individual an effective amount of a monoclonal antibody that modulates the CGRP pathway (e.g., an anti-CGRP antagonist antibody or an anti-CGRP receptor antibody) in combination with at least one additional agent useful for treating the PIH. Such additional agents include analgesic (e.g., NSAIDs) and/or anti-headache medications (e.g., triptans).

Administration of an antibody that modulates the CGRP pathway (e.g., an anti-CGRP antagonist antibody or an anti-CGRP receptor antibody) can be by any means known in the art, including: orally, intravenously, subcutaneously, intraarterially, intramuscularly, intranasally (e.g., with or without inhalation), intracardially, intraspinally, intrathoracically, intraperitoneally, intraventricularly, sublingually, transdermally, and/or via inhalation. Administration may be systemic, e.g., intravenously, or localized. In some embodiments, an initial dose and one or more additional doses are administered via the same route, i.e., subcutaneously or intravenously. In some embodiments, the one or more additional doses are administered in a different route than the initial dose, i.e., the initial dose may be administered intravenously and the one or more additional doses may be administered subcutaneously.

In some embodiments, the antibody that modulates the CGRP pathway (e.g., an anti-CGRP antagonist antibody or an anti-CGRP receptor antibody) may be administered in conjunction with another agent, such as another agent for treating PIH, e.g., NSAIDs and/or triptans.

In another aspect, the invention provides use of an antibody that modulates the CGRP pathway (e.g., an anti-CGRP antagonist antibody or an anti-CGRP receptor antibody) for the manufacture of a medicament for use in any of the methods described herein, for example, for preventing, treating, or reducing PIH.

In one aspect, provided herein is a monoclonal antibody that modulates the calcitonin gene related peptide (CGRP) pathway for use in the prevention of a post-ictal headache.

In some embodiments, the antibody comprises a CDR H1 as set forth in SEQ ID NO:3; a CDR H2 as set forth in SEQ ID NO:4; a CDR H3 as set forth in SEQ ID NO:5; a CDR L1 as set forth in SEQ ID NO:6; a CDR L2 as set forth in SEQ ID NO:7; and a CDR L3 as set forth in SEQ ID NO:8. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:1, and a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:2. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence as set forth in SEQ ID NO:11, and a light chain comprising the amino acid sequence as set forth in SEQ ID NO:12.

In some embodiments, the antibody comprises a CDR H1 as set forth in SEQ ID NO: 87; a CDR H2 as set forth in SEQ ID NO:88; a CDR H3 as set forth in SEQ ID NO:89; a CDR L1 as set forth in SEQ ID NO:84; a CDR L2 as set forth in SEQ ID NO:85; and a CDR L3 as set forth in SEQ ID NO:86. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:82, and a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:80. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence as set forth in SEQ ID NO:83, and a light chain comprising the amino acid sequence as set forth in SEQ ID NO:81.

In some embodiments, the antibody comprises a CDR H1 as set forth in SEQ ID NO:93; a CDR H2 as set forth in SEQ ID NO:94; a CDR H3 as set forth in SEQ ID NO:95; a CDR L1 as set forth in SEQ ID NO:91; a CDR L2 as set forth in SEQ ID NO:92; and a CDR L3 as set forth in SEQ ID NO:90. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:97, and a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:96. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence as set forth in SEQ ID NO:99, and a light chain comprising the amino acid sequence as set forth in SEQ ID NO:98.

In some embodiments, the antibody comprises a CDR H1 as set forth in SEQ ID NO:103; a CDR H2 as set forth in SEQ ID NO:104; a CDR H3 as set forth in SEQ ID NO:105; a CDR L1 as set forth in SEQ ID NO:100; a CDR L2 as set forth in SEQ ID NO:101; and a CDR L3 as set forth in SEQ ID NO:102. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:107, and a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:106. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence as set forth in SEQ ID NO:109, and a light chain comprising the amino acid sequence as set forth in SEQ ID NO:108.

In another aspect, the invention provides a pharmaceutical composition for preventing, treating, or reducing PIH comprising an effective amount of a monoclonal antibody that modulates the CGRP pathway (e.g., an anti-CGRP antagonist antibody or an anti-CGRP receptor antibody), in combination with one or more pharmaceutically acceptable excipients.

In another aspect, the invention provides kits for use in any of the methods described herein. In some embodiments, the kit comprises a container, a composition comprising an antibody that modulates the CGRP pathway (e.g., an anti-CGRP antagonist antibody or an anti-CGRP receptor antibody), in combination with a pharmaceutically acceptable carrier, and instructions for using the composition in any of the methods described herein.

In one aspect, the invention provides a monoclonal antibody that modulates the CGRP pathway, or a composition comprising a monoclonal antibody that modulates the CGRP pathway, for use in decreasing a number of monthly headache or migraine hours experienced by a subject. In some embodiments, the monoclonal antibody or composition is administered to the subject in an amount effective to decrease the number of monthly headache or migraine hours by at least 20 (e.g., 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or more headache or migraine hours) after a single dose. In some embodiments, the number of monthly headache or migraine hours is reduced by at least about 50 hours. In some embodiments, the monoclonal antibody or composition is administered to the subject in an amount effective to decrease the number of monthly headache or migraine hours by at least 15% (e.g., 20%, 25%, 30%, 35%, 40%, or more) after a single dose. In some embodiments, the number of monthly headache or migraine hours is reduced by at least about 30%.

In one aspect, the invention provides a monoclonal antibody that modulates the CGRP pathway, or a composition comprising a monoclonal antibody that modulates the CGRP pathway, for use in decreasing a number of monthly headache or migraine days experienced by a subject. In some embodiments, the monoclonal antibody or composition is administered to the subject in an amount effective to decrease the number of monthly headache or migraine days by at least 3 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more headache or migraine days) after a single dose. In some embodiments, the number of monthly headache or migraine days is reduced by at least about 6 headache or migraine days.

In one aspect, the invention provides a monoclonal antibody that modulates the CGRP pathway, or a composition comprising a monoclonal antibody that modulates the CGRP pathway, for use in decreasing use of any acute headache medication in a subject, comprising administering to the subject a monoclonal antibody (e.g., anti-CGRP antagonist antibody or an anti-CGRP receptor antibody) in an amount effective to decrease monthly use of the acute headache medication by the subject by at least 15% (e.g., 20%, 25%, 30%, 35%, 40%, or more). In some embodiments, the anti-headache medication is selected from the group consisting of NSAIDs and/or triptans. In some embodiments, the anti-headache medication is a triptan.

Compositions for use according to any of the methods described herein are also provided.

In one aspect, the invention provides a monoclonal antibody that modulates the CGRP pathway, or a composition comprising a monoclonal antibody that modulates the CGRP pathway, for use in preventing, treating, or reducing incidence of PIH in a subject, comprising administering to the subject a single dose of the monoclonal antibody (e.g., monoclonal anti-CGRP-antagonist antibody) in an amount that modulates the CGRP pathway, wherein the amount of the monoclonal antibody administered to the subject is about 675 mg to about 1000 mg.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table showing binding affinities of 12 murine antibodies for different alanine substituted human α-CGRP fragments. Binding affinities were measured at 25° C. using Biacore by flowing Fabs across CGRPs on the chip. The boxed values represent the loss in affinity of alanine mutants relative to parental fragment, 25-37 (italic), except K35A, which was derived from a 19-37 parent. “^(a)” indicates affinities for 19-37 and 25-37 fragments are the mean average±standard deviation of two independent measurements on different sensor chips. “^(b)” indicates these interactions deviated from a simple bimolecular interaction model due to a biphasic off rate, so their affinities were determined using a conformational change model. Grey-scale key: white (1.0) indicates parental affinity; light grey (less than 0.5) indicates higher affinity than parent; dark grey (more than 2) indicates lower affinity than parent; and black indicates that no binding was detected.

FIGS. 2A and 2B show the effect of administering CGRP 8-37 (400 nmol/kg), antibody 4901 (25 mg/kg), and antibody 7D11 (25 mg/kg) on skin blood flow measured as blood cell flux after electrical pulse stimulation for 30 seconds. CGRP 8-37 was administered intravenously (iv) 3-5 min before electrical pulse stimulation. Antibodies were administered intraperitoneal (IP) 72 hours before electrical pulse stimulation. Each point in the graphs represents AUC of one rat treated under the conditions as indicated. Each line in the graphs represents average AUC of rats treated under the condition as indicated. AUC (area under the curve) equals to Δflux×Δtime. “Δflux” represents the change of flux units after the electrical pulse stimulation; and “Δtime” represents the time period taken for the blood cell flux level to return to the level before the electrical pulse stimulation.

FIG. 3 shows the effect of administering different dosage of antibody 4901 (25 mg/kg, 5 mg/kg, 2.5 mg/kg, or 1 mg/kg) on skin blood flow measured as blood cell flux after electrical pulse stimulation for 30 seconds. Antibodies were administered intravenously (IV) 24 hours before electrical pulse stimulation. Each point in the graph represents AUC of one rat treated under the conditions as indicated. The line in the graph represents average AUC of rats treated under the condition as indicated.

FIGS. 4A and 4B show the effect of administering antibody 4901 (1 mg/kg or 10 mg/kg, i.v.), antibody 7E9 (10 mg/kg, i.v.), and antibody 8B6 (10 mg/kg, i.v.) on skin blood flow measured as blood cell flux after electrical pulse stimulation for 30 seconds. Antibodies were administered intravenously (i.v.) followed by electrical pulse stimulation at 30 min, 60 min, 90 min, and 120 min after antibody administration. Y axis represents percent of AUC as compared to level of AUC when no antibody was administered (time 0). X axis represents time (minutes) period between the administration of antibodies and electrical pulse stimulation. “*” indicates P<0.05, and “**” indicates P<0.01, as compared to time 0. Data were analyzed using one-way ANOVA with a Dunnett's Multiple comparison test.

FIG. 5 shows the amino acid sequence of the heavy chain variable region (SEQ ID NO:1) and light chain variable region (SEQ ID NO:2) of antibody G1. The Kabat CDRs are in bold text, and the Chothia CDRs are underlined. The amino acid residues for the heavy chain and light chain variable region are numbered sequentially.

FIG. 6 shows epitope mapping of antibody G1 by peptide competition using Biacore. N-biotinylated human α-CGRP was captured on SA sensor chip. G1 Fab (50 nM) in the absence of a competing peptide or pre-incubated for 1 hour with 10 μM of a competing peptide was flowed onto the chip. Binding of G1 Fab to the human α-CGRP on the chip was measured. Y axis represents percentage of binding blocked by the presence of the competing peptide compared with the binding in the absence of the competing peptide.

FIG. 7A shows how single-unit recordings were obtained from dural primary afferent nociceptors in the trigeminal ganglion while recording electrocorticogram activity from the caudal cortex. Triangle shows site of picrotoxin administration.

FIG. 7B shows dural afferents were identified by their response to single-shock stimulation applied to the dura overlying the transverse sinus, and were further characterized as mechanosensitive by their response to von Frey (VFH) stimulation of the dura.

FIG. 7C is a bar graph that shows location of dural receptive field.

FIG. 7D is an electrocorticogram (upper trace) and firing rate of a dural afferent (lower trace) before and after induction of seizure by picrotoxin. Triangle shows time of picrotoxin administration.

FIG. 8A shows an experimental setup showing locations of ECG recording in the parietal cortex, neuronal recording in lamina I of the upper cervical dorsal horn, and the neuron's dural and facial receptive fields.

FIG. 8B is a graph showing electrical stimulation on dura.

FIG. 8C is a bar graph that shows mechanical stimulation on dura.

FIG. 8D shows an ECG recording of a neuron characterized as wide dynamic range (WDR) by its responses to graded mechanical stimulation of the facial skin.

FIG. 8E shows that topical application of picrotoxin to the parietal cortex induced cortical seizure (upper trace) and transient suppression of neuronal firing, which was followed by a prolonged increase above baseline that persisted after the cessation of seizure activity.

FIG. 9 shows that an anti-CGRP antagonist antibody prevents activation of central trigeminovascular neuron by seizure. When given intravenously (30 mg/kg) four hours before induction of seizure, TEV-48125 prevented the activation of a central trigeminovascular neuron but not the occurrence of a seizure. Top panel shows the seizure activity in the cortex. Bottom panel shows lack of activity in the central neuron.

FIGS. 10A, 10B, 10C, 10D, 10E, 10F, 10G, 10H, 10I, and 10J show recording sites (FIGS. 10A and 10F), facial receptive fields (FIGS. 10B, 10D, 10G, and 10I), and dural receptive fields (FIGS. 10C, 10E, 10H, and 10J) of each of the 63 trigeminovascular neurons tested for effects of the CGRP-mAb (FIGS. 10A, 10B, 10C, 10F, 10G, and 10H, n=36) or the isotype-conAb (FIGS. 10D, 10E, 10I, and 10J, n=27) in male and female rats. FIGS. 10A and 10F show recording sites plotted on a representative transverse section through the first cervical segment. The circles represent HT and WDR neurons, as indicated. FIGS. 10B, 10D, 10G, and 10I show the most sensitive regions of cutaneous (i.e., where brush, pressure and pinch were applied) and corneal receptive fields. FIGS. 10C, 10E, 10H, and 10J show the mechanically sensitive receptive fields on the dura, which were all on or around the transverse sinus. The portion of the dura shown in the receptive field drawings is outlined by the dashed line in the inset in FIG. 10H. All dural and facial receptive fields were ipsilateral to the recorded neuron. Abbreviations: HT, high-threshold; WDR, wide-dynamic range.

FIGS. 11A, 11B, 11C, 11D, 11E, 11F, and 11G show the effect of CGRP-mAb (FIGS. 11A, 11B, 11C, and 11D) and isotype-conAb (FIGS. 11E, 11F, and 11G) on the spontaneous activity of trigeminovascular neurons in male and female rats. FIGS. 11A, 11D, and 11F are plots of spontaneous discharge rate recorded at baseline (BL) and at 1-4 hours following CGRP-mAb (FIG. 11A) or isotype-conAb (FIG. 11E) administration to HT neurons. Numbers in parentheses show the mean discharge rate for the 15-minute sampling period at each time point. Bin width=1 sec. FIGS. 11B and 11C are histograms showing mean (±S.E.) spontaneous discharge of HT and WDR neurons recorded at baseline and 1-4 hours following CGRP-mAb administration in male (FIG. 11B) and female (FIG. 11C) rats. FIGS. 11F and 11G are histograms showing mean (±S.E.) spontaneous discharge of HT and WDR neurons recorded at baseline and 1-4 hours following isotype-conAb administration in male (FIG. 11F) and female (FIG. 11G) rats. * p<0.05 compared to baseline. Numbers in parentheses in FIGS. 11B, 11C, 11F, and 11G depict the number of neurons in each group.

FIGS. 12A, 12B, 12C, 12D, 12E, and 12F are graphs showing the effect of CGRP-mAb (FIGS. 12A, 12B, and 12C) and isotype-conAb (FIGS. 12D, 12E, and 12F) on the response of trigeminovascular neurons to dural indentation in male and female rats. FIGS. 12A and 12D are graphs showing the responses to indentation of the dura with a von Frey hair (VFH, 4.19 g) at baseline (BL) and at 1-4 hours following CGRP-mAb (FIG. 12A) or isotype control antibody (isotype-conAb) (FIG. 12D) administration to HT neurons. Numbers in parentheses show the mean discharge rate during the stimulus. Bin width=1 sec. FIGS. 12B and 12C are graphs showing the mean (±S.E.) discharge rates in response to dural stimulation at baseline and 1-4 hours following drug administration for the entire sample of neurons that received CGRP-mAb in male (FIG. 12B) and female (FIG. 12C) rats. FIGS. 12E and 12F are graphs showing the mean (±S.E.) discharge rates in response to dural stimulation at baseline and 1-4 hours following drug administration for the entire sample of neurons that received isotype-conAb in male (FIG. 12E) and female (FIG. 12F) rats. * p<0.05 compared to baseline. Numbers in parentheses in FIGS. 12B, 12C, 12E, and 12F depict number of neurons in each group.

FIGS. 13A, 13B, 13C, 13D, 13E, 13F, 13G, and 13H are graphs showing the effect of CGRP-mAb (FIGS. 13A, 13B, 13C, 13D) and isotype-conAb (FIGS. 13E, 13F, 13G, and 13H) on the response of central trigeminovascular neurons to innocuous and noxious mechanical stimulation of cutaneous receptive fields of male and female rats. FIGS. 13A, 13B, 13E, and 13F are graphs showing the responses to mechanical stimulation of the cutaneous receptive fields of HT (FIGS. 13A and 13E) and WDR (FIGS. 13B and 13F) with brush, pressure, and pinch, at baseline (BL) and at 1-4 hours following CGRP-mAb or isotype-conAb administration. Numbers in parentheses show the mean discharge rate during each stimulus. Bin width=1 sec. FIGS. 13C and 13D are graphs showing the mean (±S.E.) discharge rates in response to cutaneous stimulation at baseline and 1-4 hours following drug administration for the entire sample of neurons that received CGRP-mAb in male (FIG. 13C) and female (FIG. 13D) rats. FIGS. 13G and 13H are graphs showing the mean (±S.E.) discharge rates in response to cutaneous stimulation at baseline and 1-4 hours following drug administration for the entire sample of neurons that received isotype-conAb in male (FIG. 13G) and female (FIG. 13H) rats. Numbers in parentheses in FIGS. 13C, 13D, 13G, and 13H depict number of neurons in each group.

FIGS. 14A, 14B, 14C, 14D, 14E, and 14F are graphs showing the effect of CGRP-mAb (FIGS. 14A, 14B, and 14C) and isotype-conAb (FIGS. 14D, 14E, and 14F) on the response of central trigeminovascular neurons to mechanical stimulation of the cornea in male and female rats. FIGS. 14A and 14D are graphs showing the responses to mechanical stimulation of the cornea by gentle brushing, at baseline (BL) and at 1-4 hours following CGRP-mAb (FIG. 14A) or isotype-conAb (FIG. 14D) administration to HT neurons. Numbers in parentheses show the mean discharge rate during each stimulus. Bin width=1 sec. FIGS. 14B and 14C are graphs showing the mean (±S.E.) discharge rates in response to cornea stimulation at baseline and 1-4 hours following drug administration for the entire sample of neurons that received CGRP-mAb in male (FIG. 14B) and female (FIG. 14C) rats. FIGS. 14E and 14F are graphs showing the mean (±S.E.) discharge rates in response to cornea stimulation at baseline and 1-4 hours following drug administration for the entire sample of neurons that received isotype-conAb in male (FIG. 14E) and female (FIG. 14F) rats. Numbers in parentheses in FIGS. 14B, 14C, 14E, and 14F depict number of neurons in each group.

FIGS. 15A, 15B, 15C, 15D, 15E, and 15F are graphs showing the effect of CGRP-mAb (FIGS. 15A, 15B, and 15C) and isotype-conAb (FIGS. 15D, 15E, and 15F) on the activation of trigeminovascular neurons by cortical spreading depression (CSD) induced 4 hours post-drug treatment. FIGS. 15A and 15D are graphs show the discharge of trigeminovascular neurons prior to CSD induction (top), during CSD induction (middle), and 2 hours post-CSD (bottom), in two HT neurons that received CGRP-mAb (FIG. 15A) or isotype-conAb (FIG. 15D) 4 hours before CSD induction. Bin width=1 sec. FIGS. 15B and 15C are graphs showing the mean (±S.E.) discharge rates for the entire sample of HT and WDR trigeminovascular neurons tested for CSD responses after isotype-conAb administration in male (FIG. 15B) and female (FIG. 15C) rats. FIGS. 15E and 15F are graphs showing the mean (±S.E.) discharge rates for the entire sample of HT and WDR trigeminovascular neurons tested for CSD responses after CGRP-mAb administration in male (FIG. 15E) and female (FIG. 15F) rats. Discharge is shown at baseline (4 hours post-drug treatment, prior to CSD induction) and 2 hours post-CSD. * p<0.05 compared to baseline. Numbers in parentheses in FIGS. 15B, 15C, 15E, and 15F depict number of neurons in each group.

FIGS. 16A and 16B depict the expansion of dural and cutaneous receptive fields following occurrence of CSD in male and female rats. Blue (upper left to lower right diagonal lines) and pink (upper right to lower left diagonal lines) illustrate dural and cutaneous receptive fields before, and 2 hours after CSD induction, respectively, in isotype-conAb (FIG. 16A) and CGRP-mAb (FIG. 16B) treated rats.

FIGS. 17A, 17B, 17C, 17D, 17E, and 17F are graphs showing that the enhanced responses to mechanical stimulation of the dura following CSD are prevented by the CGRP-mAb. FIGS. 17A and 17D are graphs showing the responses to indentation of the dura prior to CSD induction (BL) and 2 hours post-CSD, in two HT neurons that received treatment with CGRP-mAb (FIG. 17A) or isotype-conAb (FIG. 17D) 4 hours prior to CSD induction. Numbers in parentheses show the mean discharge rate during each stimulus. Bin width=1 sec. FIGS. 17B, 17C, 17E, and 17F are graphs showing the mean (±S.E.) discharge in response to dural indentation prior to CSD induction (Baseline) and 2 hours post-CSD, in neurons that received treatment with isotype-conAb (FIGS. 17B and 17C) or CGRP-mAb (FIGS. 17E and 17F). Neurons recorded in males are shown in FIGS. 17B and 17E; neurons recorded in females are shown in FIGS. 17C and 17F. * p<0.05 compared to baseline. Numbers in parentheses in FIGS. 17B, 17C, 17E, and 17F depict number of neurons in each group.

FIGS. 18A, 18B, 18C, 18D, 18E, and 18F are graphs showing the enhanced responses to cutaneous stimulation following CSD are prevented by the CGRP-mAb. FIGS. 18A and 18D are graphs showing the responses to mechanical stimulation of the cutaneous receptive fields with brush, pressure, and pinch, prior to CSD induction (BL) and 2 hours post-CSD, for two HT neurons that received CGRP-mAb (FIG. 18A) or isotype-conAb (FIG. 18D) 4 hours before CSD induction. Numbers in parentheses show the mean discharge rate during each stimulus. Bin width=1 sec. FIGS. 18B, 18C, 18E, and 18F are graphs showing the mean (±S.E.) discharge in response to cutaneous stimulation prior to (Baseline) and 2 hours post-CSD induction, in HT neurons that received treatment with isotype-conAb or CGRP-mAb 4 hours prior to CSD induction. Neurons recorded in males are shown in FIGS. 18B and 18E; neurons recorded in females are shown in FIGS. 18C and 18F. * p<0.05 compared to baseline.

FIGS. 19A, 19B, 19C, 19D, 19E, and 19F are graphs showing the enhanced responses to mechanical stimulation of the cornea following CSD are prevented by CGRP-mAb (female only). FIGS. 19A and 19D are graphs showing the responses to corneal stimulation (gentle brush) prior to CSD induction (BL) and 2 hours post-CSD, in two HT neurons that received treatment with isotype-conAb (FIG. 19A) or CGRP-mAb (FIG. 19D) 4 hours prior to CSD induction. Bin width=1 sec. FIGS. 19B, 19C, 19E, and 19F are graphs showing the mean (±S.E.) discharge in response to corneal stimulation prior to CSD induction (Baseline) and 2 hours post-CSD, in neurons that received treatment with isotype-conAb (FIGS. 19B and 19C) or CGRP-mAb (FIGS. 19E and 19F) 4 hours prior to CSD induction. Neurons recorded in males are shown in FIGS. 19B and 19E; neurons recorded in females are shown in FIGS. 19C and 19F. * p<0.05 compared to baseline.

FIG. 20 are tables showing the results of the studies (as described in Example 6) of spontaneous activity of the HT and WDR neurons in male and female rats in a naïve state and post-CSD state upon application of the indicated stimuli.

FIGS. 21A, 21B and 21C are graphs showing the activation of a-delta meningeal nociceptors by CSD. FIG. 21A are graphs showing an exemplary individual a-delta fiber response to CSD. The baseline spontaneous activity of the neuron is shown from 0 to 60 min whereas the firing rate of the neuron after CSD is shown from 60 to 120 min. FIG. 21B is a bar graph showing the mean (±SE) response magnitude of the six a-delta fibers that were activated by CSD (p<0.05). FIG. 21C is a graph showing the changes in response frequency of all six a-delta neurons.

FIGS. 22A, 22B, and 22C are graphs showing the activation of C-type meningeal nociceptors by CSD. FIG. 22A are graphs showing an exemplary individual C-type fiber response to CSD. The baseline spontaneous activity of the neuron is shown from 0 to 60 min whereas the firing rate of the neuron after CSD is shown from 60 to 120 min. FIG. 22B shows the mean (±SE) response magnitude of the six C-type fibers that were activated by CSD (p<0.05). FIG. 22C shows the changes in response frequency of all six C-type neurons.

FIGS. 23A and 23B show that fremanezumab prevents the activation of most a-delta and some C-type meningeal nociceptors. FIG. 23A shows an individual example of fremanezumab treated a-delta fiber showing no change in spontaneous activity after CSD. FIG. 23B shows examples of responses of two C-type meningeal nociceptors to CSD after treatment with fremanezumab. Note that the upper neuron was not activated by CSD whereas the lower neuron was activated by CSD.

FIGS. 24A and 24B are tables showing the incidence of activation of A-delta or C-type meningeal nociceptors by CSD.

DETAILED DESCRIPTION

In some aspects, the invention disclosed herein provides methods for preventing, treating, and/or reducing PIH in an individual by administering to the individual a therapeutically effective amount of a monoclonal antibody that modulates the CGRP pathway (e.g., an anti-CGRP antagonist antibody or an anti-CGRP receptor antibody).

General Techniques

The practice of the various aspects of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995).

Definitions

As used herein, “about” when used in reference to numerical ranges, cutoffs, or specific values is used to indicate that the recited values may vary by up to as much as 10% from the listed value. Thus, the term “about” is used to encompass variations of ±10% or less, variations of ±5% or less, variations of ±1% or less, variations of ±0.5% or less, or variations of ±0.1% or less from the specified value.

An “antibody” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab′, F(ab′)₂, Fv), single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion (such as domain antibodies), and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

As used herein, “monoclonal antibody” or “mAb” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature, 256:495, or may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature, 348:552-554, for example.

As used herein, “humanized” antibodies refer to forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and, biological activity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Antibodies may have Fc regions modified as described in WO 99/58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody.

As used herein, “human antibody” means an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies known in the art or disclosed herein. This definition of a human antibody includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising murine light chain and human heavy chain polypeptides. Human antibodies can be produced using various techniques known in the art. In one embodiment, the human antibody is selected from a phage library, where that phage library expresses human antibodies (Vaughan et al., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, PNAS, (USA) 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol., 222:581). Human antibodies can also be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016. Alternatively, the human antibody may be prepared by immortalizing human B lymphocytes that produce an antibody directed against a target antigen (such B lymphocytes may be recovered from an individual or may have been immunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., 1991, J. Immunol., 147 (1):86-95; and U.S. Pat. No. 5,750,373.

As used herein, the term “calcitonin gene-related peptide” and “CGRP” refers to any form of calcitonin gene-related peptide and variants thereof that retain at least part of the activity of CGRP. For example, CGRP may be α-CGRP or β-CGRP. As used herein, CGRP includes all mammalian species of native sequence CGRP, e.g., human, canine, feline, equine, and bovine.

As used herein, an “anti-CGRP antibody” refers to an antibody that modulates CGRP biological activity, or the CGRP pathway, including downstream pathways mediated by CGRP signaling, such as receptor binding and/or elicitation of a cellular response to CGRP. For example, an anti-CGRP antibody may block, inhibit, suppress or reduce the calcitonin gene related peptide (CGRP) pathway. The term anti-CGRP antibody encompasses both “anti-CGRP antagonist antibodies” and “anti-CGRP receptor antibodies.” In some embodiments, the anti-CGRP antibody is a monoclonal antibody (i.e., an anti-CGRP monoclonal antibody).

As used herein, an “anti-CGRP antagonist antibody” refers to an antibody that is able to bind to CGRP and inhibit CGRP biological activity and/or downstream pathway(s) mediated by CGRP signaling. An anti-CGRP antagonist antibody encompasses antibodies that modulate, block, antagonize, suppress or reduce (including significantly) CGRP biological activity, or otherwise antagonize the CGRP pathway, including downstream pathways mediated by CGRP signaling, such as receptor binding and/or elicitation of a cellular response to CGRP. For purpose of the present invention, it will be explicitly understood that the term “anti-CGRP antagonist antibody” encompasses all the previously identified terms, titles, and functional states and characteristics whereby CGRP itself, CGRP biological activity (including but not limited to its ability to mediate any aspect of headache), or the consequences of the biological activity, are substantially nullified, decreased, or neutralized in any meaningful degree. In some embodiments, an anti-CGRP antagonist antibody binds CGRP and prevents CGRP binding to a CGRP receptor. In other embodiments, an anti-CGRP antagonist antibody binds CGRP and prevents activation of a CGRP receptor. Examples of anti-CGRP antagonist antibodies are provided herein.

An “anti-CGRP receptor antibody” refers to an antibody that is able to bind to a CGRP receptor and thereby modulate the CGRP pathway. Examples of anti-CGRP receptor antibodies are provided herein (e.g., erenumab).

As used herein, the terms “G1,” “antibody G1,” “TEV-48125” and “fremanezumab” are used interchangeably to refer to an anti-CGRP antagonist antibody produced by expression vectors having deposit numbers of ATCC PTA-6867 and ATCC PTA-6866. The amino acid sequence of the heavy chain and light chain variable regions are shown in FIG. 5. The CDR portions of antibody G1 (including Chothia and Kabat CDRs) are diagrammatically depicted in FIG. 5. The polynucleotides encoding the heavy and light chain variable regions are shown in SEQ ID NO:9 and SEQ ID NO:10. The G1 heavy chain full length amino acid sequence is shown in SEQ ID NO:11. The G1 light chain full length amino acid sequence is shown in SEQ ID NO:12. The characterization of G1 is described in the Examples, as well as PCT Publication No. WO 2007/054809 and WHO Drug Information 30(2): 280-1 (2016), which are hereby incorporated by reference in its entirety.

The terms “ALD403,” and “eptinezumab” refer to an anti-CGRP antagonist antibody, which is a humanized IgG1 monoclonal antibody from a rabbit precursor. Characterization and processes for making eptinezumab can be found in U.S. Publication No. US 2012/0294797 and WHO Drug Information 30(2): 274-5 (2016), which are incorporated by reference in its entirety.

The terms “LY2951742,” and “galcanezumab” refer to an anti-CGRP antagonist antibody, which is a humanized IgG4 monoclonal antibody from a murine precursor. Characterization and processes for making galcanezumab can be found in U.S. Publication No. US 2011/0305711 and WHO Drug Information 29(4): 526-7 (2015), which is incorporated by reference in its entirety. Dosing and formulations associated with galcanezumab can be found in PCT Publication No. WO 2016/205037, which is also incorporated by reference in its entirety.

The terms “AMG334,” and “erenumab” refer to an anti-CGRP receptor antibody, which is a fully humanized IgG2 antibody. Characterization and processes for making erenumab can be found in U.S. Publication No. US 2010/0172895 and U.S. Pat. No. 9,102,731, and WHO Drug Information 30(2): 275-6 (2016), each of which are incorporated by reference in their entireties. Dosing and formulations associated with erenumab can be found in PCT Publication No. WO 2016/171742, which is also incorporated by reference in its entirety.

The terms “polypeptide”, “oligopeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that, because the polypeptides of this invention are based upon an antibody, the polypeptides can occur as single chains or associated chains.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports. The 5′ and 3′ terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), (O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂ (“formacetal”), in which each R or R′ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

As used herein, a “post-ictal headache” (PIH) can be a headache that is attributed to a non-vascular intracranial disorder and can be caused by and occurring within three hours after an epileptic seizure, and remitting spontaneously within 72 hours after seizure termination, as further described in The International Classification of Headache Disorders, 3rd edition (beta version), Cephalalgia, 33(9): 629-808 (2013). PIH occurs in over 40% of subjects with either temporal lobe epilepsy or frontal lobe epilepsy and in up to 60% of subjects with occipital lobe epilepsy. It occurs more frequently after generalized tonic-clonic seizures than other seizure types.

Skilled practitioners will be readily able to recognize a subject with a PIH. For example, diagnostic criteria for a PIH can include:

A. Any headache fulfilling criterion C

B. The patient has recently had a partial or generalized epileptic seizure

C. Evidence of causation demonstrated by both of the following:

-   -   1. Headache has developed within three hours after the epileptic         seizure has terminated     -   2. Headache has resolved within 72 hours after the epileptic         seizure has terminated

D. Not better accounted for by another ICHD-3 diagnosis.

As used herein, “preventing” is an approach to stop PIH from occurring or existing in a subject, who does not already have PIH. As used herein, “treatment” is an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: improvement in any aspect of a PIH, including lessening severity, alleviation of pain intensity, and other associated symptoms, increasing the quality of life of those suffering from the PIH, and decreasing dose of other medications required to treat the PIH. The terms “patient” and “subject” are used interchangeably herein. In some embodiments, the patient is a human.

“Reducing incidence” of PIH means any of reducing severity (which can include reducing need for and/or amount of (e.g., exposure to) other drugs and/or therapies generally used for this condition, including, for example, NSAIDS and/or triptans) and/or duration. As is understood by those skilled in the art, individuals may vary in terms of their response to treatment, and, as such, for example, a “method of reducing incidence of PIH in an individual” reflects administering an anti-CGRP antagonist antibody or an anti-CGRP receptor antibody based on a reasonable expectation that such administration may likely cause such a reduction in incidence in that particular individual.

“Ameliorating” PIH or one or more symptoms of PIH means a lessening or improvement of one or more symptoms of PIH as compared to not administering an anti-CGRP antagonist antibody or an anti-CGRP receptor antibody. “Ameliorating” also includes shortening or reduction in duration of a symptom.

As used herein, “controlling PIH” refers to maintaining or reducing severity or duration of one or more symptoms of PIH in an individual (as compared to the level before treatment). For example, the duration or severity of head pain is reduced by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, in the individual as compared to the level before treatment.

As explained above, for non-migrainous PIHs, the relevant endpoint is a reduction in headache hours after a seizure. For migrainous PIHs, a reduction in headache hours, headache days, migraine hours, migraine days (per day or per month) can be relevant. Therefore, “migraine hours” and “migraine days” are used herein only in the context of PIHs that are migrainous.

As used herein, a “headache hour” and a “migraine hour” refers to an hour during which a subject experiences headache or migraine, respectively. Headache or migraine hours can be expressed in terms of whole hours (e.g., one headache or migraine hour, two headache or migraine hours, three headache or migraine hours, etc.) or in terms of whole and partial hours (e.g., 0.5 headache or migraine hours, 1.2 headache or migraine hours, 2.67 headache or migraine hours, etc.). One or more headache or migraine hours may be described with respect to a particular time interval. For example, “daily headache or migraine hours” may refer to the number of headache or migraine hours a subject experiences within a day interval (e.g., a 24-hour period). In another example, “weekly headache or migraine hours” may refer to the number of headache or migraine hours a subject experiences within a week interval (e.g., a 7-day period). As can be appreciated, a week interval may or may not correspond to a calendar week. In another example, “monthly headache or migraine hours” may refer to the number of headache or migraine hours a subject experiences within a month interval. As can be appreciated, a month interval (e.g., a period of 28, 29, 30, or 31 days) may vary in terms of number of days depending upon the particular month and may or may not correspond to a calendar month.

As used herein, a “headache day” and a “migraine day” refers to a day during which a subject experiences headache or migraine, respectively. Headache or migraine days can be expressed in terms of whole days (e.g., one headache or migraine day, two headache or migraine days, three headache or migraine days, etc.) or in terms of whole and partial days (e.g., 0.5 headache or migraine days, 1.2 headache or migraine days, 2.67 headache or migraine days, etc.). One or more headache or migraine days may be described with respect to a particular time interval. For example, “weekly headache or migraine days” may refer to the number of headache or migraine days a subject experiences within a week interval (e.g., a 7-day period). As can be appreciated, a week interval may or may not correspond to a calendar week. In another example, “monthly headache or migraine days” may refer to the number of headache or migraine days a subject experiences within a month interval. As can be appreciated, a month interval (e.g., a period of 28, 29, 30, or 31 days) may vary in terms of number of days depending upon the particular month and may or may not correspond to a calendar month.

As used therein, “delaying” the development of PIH means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop PIH. A method that “delays” development of the symptom is a method that reduces probability of developing the symptom in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a statistically significant number of subjects.

“Development” or “progression” of PIH means initial manifestations and/or ensuing progression of the disorder. Development of PIH can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence and onset. As used herein “onset” or “occurrence” of PIH includes initial onset.

As used herein, an “effective dosage” or “effective amount” of drug, compound, or pharmaceutical composition is an amount sufficient to effect beneficial or desired results. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk, lessening the severity, or delaying the onset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing pain intensity or duration, and decreasing one or more symptoms resulting from PIH (biochemical, histological and/or behavioral), including its complications and intermediate pathological phenotypes presenting during development of the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication, and/or delaying the progression of the disease of patients. An effective dosage can be administered in one or more administrations. For purposes of this disclosure, an effective dosage of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “effective dosage” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.

An “individual” or a “subject” is a mammal, more preferably a human. Mammals also include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats.

As used herein, a “subject who is at risk for a PIH” may have a brain infection, traumatic brain injury, personal history of a seizure, family history of epilepsy, stroke, and/or dementia. Skilled practitioners will be able to ascertain subjects who are at risk for a PIH.

A. Methods for Preventing, Treating, or Reducing PIH and/or at Least One Secondary Symptom Associated with Migrainous PIH

In one aspect, the invention provides methods of preventing, treating, or reducing incidence of PIH, e.g., migrainous PIH and non-migrainous PIH, in a subject. In another aspect, the invention provides a method of preventing, treating, or reducing incidence of at least one secondary symptom associated with migrainous PIH in a subject. In some embodiments, the method comprises administering to the individual an effective amount of an antibody or polypeptides derived from the antibody that modulates the CGRP pathway (e.g., a monoclonal anti-CGRP antagonist antibody or a monoclonal anti-CGRP receptor antibody).

In another aspect, the invention provides methods for preventing, ameliorating, controlling, reducing severity of, reducing incidence of, or delaying the development or progression of PIH in an individual or symptoms associated with PIH comprising administering to the individual an effective amount of an antibody that modulates the CGRP pathway (e.g., an anti-CGRP antagonist antibody or an anti-CGRP receptor antibody) in combination with at least one additional agent useful for preventing, treating, or reducing PIH. In some instances, the at least one additional agent can be administered simultaneously or sequentially with the antibody. In some embodiments, a therapeutic effect may be greater as compared to use of an antibody or one or more additional agent(s) alone. Accordingly, a synergistic effect between an antibody and the one or more additional agents may be achieved. In some embodiments, the one or more additional agent(s) may be taken by a subject prophylactically.

Such additional agents include, but are not limited to, NSAIDs and triptans. For example, the antibody and the at least one additional agent can be concomitantly administered, i.e., they can be given in close enough temporal proximity to allow their individual therapeutic effects to overlap. For example, the amount of NSAID administered in combination with an antibody that modulates the CGRP pathway (e.g., an anti-CGRP antibody) should be sufficient to produce longer lasting efficacy compared to the administration of either one of these agents in the absence of the other.

Additional non-limiting examples of additional agents that may be administered in combination with an antibody that modulates the CGRP pathway (e.g., an anti-CGRP antagonist antibody or an anti-CGRP receptor antibody) include triptans and/or a 5 hydroxytryptamine 1F receptor agonist (i.e., a serotonin receptor agonist) and/or a nonsteroidal anti-inflammatory drug (NSAID), e.g., aspirin, diclofenac, diflusinal, etodolac, fenbufen, fenoprofen, flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac, tolmetin or zomepirac, cyclooxygenase-2 (COX-2) inhibitors, celecoxib, rofecoxib, meloxicam, JTE-522, L-745,337, NS398, or a pharmaceutically acceptable salt thereof.

Non-limiting examples of triptans that can be used in combination with an antibody that modulates the CGRP pathway include sumatriptan, zolmitriptan, naratriptan, rizatriptan, eletriptan, almotriptan, and afrovatriptan.

A non-limiting example of a 5 hydroxytryptamine 1F receptor agonist is lasmiditan.

Those skilled in the art will be able to determine appropriate dosage amounts for particular agents to be used in combination with the antibody that modulates the CGRP pathway (e.g., an anti-CGRP antibody). For example, sumatriptan may be administered in a dosage from about 0.01 to about 300 mg. In some cases, sumatriptan may be administered in a dosage from about 2 mg to about 300 mg, e.g., about 5 mg to about 250 mg, about 5 mg to about 200 mg, about 5 mg to about 100 mg, about 5 mg to about 50 mg, or about 5 mg to about 25 mg. When administered non-parenterally, the typical dosage of sumatriptan is from about 25 to about 100 mg with about 50 mg being generally preferred, e.g., about 45 mg, about 55 mg, or about 60 mg. When sumatriptan is administered parenterally, the preferred dosage is about 6 mg, e.g., about 5 mg, about 7 mg, or about 8 mg. However, these dosages may be varied according to methods standard in the art so that they are optimized for a particular patient or for a particular combination therapy. Further, for example, celecoxib may be administered in an amount of between 50 and 500 mg, e.g., about 50 mg to about 400 mg, about 50 mg to about 300 mg, about 50 mg to about 200 mg, about 50 mg to about 100 mg, about 100 mg to about 400 mg, or about 200 mg to about 300 mg.

In another aspect, the disclosure provides a method of preventing, treating, or reducing incidence of PIH in a subject comprising administering to the subject a monoclonal antibody that modulates the CGRP pathway (e.g., an anti-CGRP antagonist antibody or an anti-CGRP receptor antibody).

In another aspect, the disclosure provides a method of preventing, treating, or reducing incidence of PIH in a subject comprising administering to the subject a single dose of a monoclonal antibody (e.g., a monoclonal, anti-CGRP antagonist antibody or an anti-CGRP receptor antibody) in an amount that modulates the CGRP pathway.

In another aspect, the disclosure provides a method of preventing, treating, or reducing incidence of PIH in a subject comprising administering to the subject a monthly dose of a monoclonal antibody (e.g., a monoclonal, anti-CGRP antagonist antibody or an anti-CGRP receptor antibody) in an amount that modulates the CGRP pathway.

In another aspect, the disclosure provides a method of decreasing a number of monthly headache or migraine hours experienced by a subject, comprising administering to the subject an amount of a monoclonal antibody (e.g., a monoclonal anti-CGRP antagonist antibody or a monoclonal anti-CGRP receptor antibody) that modulates the CGRP pathway. In some embodiments, the monoclonal antibody can be in an amount effective to decrease the number of monthly headache or migraine hours by at least 0.1, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more headache or migraine hours after a single dose, monthly dose, or quarterly dose. In some embodiments, the monoclonal antibody can be in an amount effective to decrease the number of monthly headache or migraine hours by at least 20 headache or migraine hours after a single dose, monthly dose, or quarterly dose. In some embodiments, the monoclonal antibody can be in an amount effective to decrease the number of monthly headache or migraine hours by at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or more headache or migraine hours. In some embodiments, the monoclonal antibody can be in an amount effective to decrease the number of monthly headache or migraine hours by at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more after a single dose. In some embodiments, the monoclonal antibody can be in an amount effective to decrease the number of monthly headache or migraine hours by at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more after a single dose, monthly dose, or quarterly dose.

In another aspect, the disclosure provides a method of decreasing a number of monthly headache or migraine days experienced by a subject, comprising administering to the subject an amount of a monoclonal antibody (e.g., a monoclonal anti-CGRP antagonist antibody or a monoclonal anti-CGRP receptor antibody) that modulates the CGRP pathway. In some embodiments, the monoclonal antibody can be in an amount effective to decrease the number of monthly headache or migraine days by at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more headache or migraine days after a single dose. In some embodiments, the monoclonal antibody can be in an amount effective to decrease the number of monthly headache or migraine days by at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more headache or migraine days after a monthly dose or quarterly dose. In some embodiments, the monoclonal antibody can be in an amount effective to decrease the number of monthly headache or migraine days by at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more after a single dose, monthly dose, or quarterly dose.

In another aspect, the disclosure provides a method of decreasing use of an anti-headache medication in a subject, comprising administering to the subject a monoclonal antibody (e.g., a monoclonal anti-CGRP antagonist antibody or a monoclonal anti-CGRP receptor antibody) that modulates the CGRP pathway. In some embodiments, the monoclonal antibody can be in an amount effective to decrease daily, monthly, quarterly, and/or yearly use of the anti-headache medication by the subject by at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more. In some embodiments, the monoclonal antibody can be in an amount effective to decrease monthly use of the anti-headache medication by the subject by at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more. The anti-headache medication can be any type of anti-headache medication described herein. Non-limiting examples of anti-headache medications include, for example, triptans (e.g., sumatriptan, zolmitriptan, naratriptan, rizatriptan, eletriptan, almotriptan, afrovatriptan), and non-steroidal anti-inflammatory drugs (NSAIDs) (e.g., aspirin, diclofenac, diflusinal, etodolac, fenbufen, fenoprofen, flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac, tolmetin or zomepirac, cyclooxygenase-2 (COX-2) inhibitors, celecoxib, rofecoxib, meloxicam, JTE-522, L-745,337, NS398, or a pharmaceutically acceptable salt thereof).

Particularly useful monoclonal antibodies for use in any of the methods described herein are anti-CGRP antibodies that include a) a CDR H1 as set forth in SEQ ID NO:3; a CDR H2 as set forth in SEQ ID NO:4; a CDR H3 as set forth in SEQ ID NO:5; a CDR L1 as set forth in SEQ ID NO:6; a CDR L2 as set forth in SEQ ID NO:7; and a CDR L3 as set forth in SEQ ID NO:8 or b) a variant of an antibody according to (a) as shown in Table 5. In some embodiments, the monoclonal antibody comprises a heavy chain variable region comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:1, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the monoclonal antibody comprises a heavy chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:11, and a light chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:12. An exemplary monoclonal antibody is fremanezumab (also referred to herein as “G1”). Monoclonal antibodies including these amino acid sequences (e.g., fremanezumab) can be administered at a dose of from about 225 mg to about 900 mg, e.g., a dose of about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 500 mg, about 525 mg, about 550 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, or about 900 mg. These does may be administered to the subject monthly or quarterly. In one exemplary treatment, the dosing regimen can include an initial dose (e.g., 675 mg), and further include administering to the subject an additional 225 mg dose of the monoclonal antibody once per month in each of the two months (or three months, four months, five months, six months, or twelve months) subsequent to the month in which the subject receives the initial dose.

Also useful in any of the methods described herein are monoclonal antibodies that include a CDR H1 as set forth in SEQ ID NO:87; a CDR H2 as set forth in SEQ ID NO:88; a CDR H3 as set forth in SEQ ID NO:89; a CDR L1 as set forth in SEQ ID NO:84; a CDR L2 as set forth in SEQ ID NO:85; and a CDR L3 as set forth in SEQ ID NO:86. In some embodiments, the monoclonal antibody comprises a heavy chain variable region comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:82, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:80. In some embodiments, the monoclonal antibody comprises a heavy chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:83, and a light chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:81. Exemplary of such an antibody would be eptinezumab. Monoclonal antibodies having these amino acid sequences (e.g., eptinezumab) may be administered at a dose of about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg. Any of the doses provided herein (e.g., about 100 mg, about 300 mg, or about 1000 mg) may be administered intravenously or subcutaneously.

Also useful in any of the methods described herein are monoclonal antibodies that include a CDR H1 as set forth in SEQ ID NO:93; a CDR H2 as set forth in SEQ ID NO:94; a CDR H3 as set forth in SEQ ID NO:95; a CDR L1 as set forth in SEQ ID NO:91; a CDR L2 as set forth in SEQ ID NO:92; and a CDR L3 as set forth in SEQ ID NO:90. In some embodiments, the monoclonal antibody comprises a heavy chain variable region comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:97, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:96. In some embodiments, the monoclonal antibody comprises a heavy chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:99, and a light chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:98. Exemplary of such an antibody would be galcanezumab. Monoclonal antibodies having these amino acid sequences (e.g., galcanezumab) may be administered at a dose of about 100 mg, about 120 mg, about 150 mg, about 200 mg, about 240 mg, about 250 mg, about 300 mg, about 350 mg, about 360 mg, about 400 mg, about 450 mg, about 480 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg. Further, the 120 mg dose may be administered in a volume of about 1.5 mL and the 240 mg dose may be administered in a volume of about 3 mL. Any of the doses provided herein (e.g., about 120 mg or about 240 mg) may be administered intravenously or subcutaneously.

Also useful in any of the methods described herein are monoclonal antibodies that include a CDR H1 as set forth in SEQ ID NO:103; a CDR H2 as set forth in SEQ ID NO:104; a CDR H3 as set forth in SEQ ID NO:105; a CDR L1 as set forth in SEQ ID NO:100; a CDR L2 as set forth in SEQ ID NO:101; and a CDR L3 as set forth in SEQ ID NO:102. In some embodiments, the monoclonal antibody comprises a heavy chain variable region comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:107, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:106. In some embodiments, the monoclonal antibody comprises a heavy chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:109, and a light chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:108. Exemplary of such an antibody would be erenumab. Monoclonal antibodies having these amino acid sequences (e.g., erenumab) may be administered at a dose of about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, or about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg. Further, the 70 mg does may be administered in a volume of about 1 mL. The 140 mg dose may be administered in a volume of about 2 mL. Any of the doses provided herein (e.g., about 70 mg or about 140 mg) may be administered intravenously or subcutaneously.

Accordingly, in certain methods described herein, a monoclonal antibody to be used in the methods described herein may be selected from the group consisting of fremanezumab, eptinezumab, galcanezumab, erenumab, and bioequivalents thereof.

In some embodiments of any of the methods described herein, a single dose of a monoclonal antibody that modulates the CGRP pathway may be an amount of antibody between 0.1 mg-5000 mg, 1 mg-5000 mg, 10 mg-5000 mg, 100 mg-5000 mg, 1000 mg-5000 mg, 0.1 mg-4000 mg, 1 mg-4000 mg, 10 mg-4000 mg, 100 mg-4000 mg, 1000 mg 4000 mg, 0.1 mg-3000 mg, 1 mg-3000 mg, 10 mg-3000 mg, 100 mg-3000 mg, 1000 mg 3000 mg, 0.1 mg-2000 mg, 1 mg-2000 mg, 10 mg-2000 mg, 100 mg-2000 mg, 1000 mg 2000 mg, 0.1 mg-1000 mg, 1 mg-1000 mg, 10 mg-1000 mg or 100 mg-1000 mg, 10 mg-3000 mg, 10 mg-2000 mg, 100 mg-2000 mg, 150 mg-2000 mg, 200 mg-2000 mg, 250 mg-2000 mg, 300 mg-2000 mg, 350 mg-2000 mg, 400 mg-2000 mg, 450 mg-2000 mg, 500 mg-2000 mg, 550 mg-2000 mg, 600 mg-2000 mg, 650 mg-2000 mg, 700 mg-2000 mg, 750 mg-2000 mg, 800 mg-2000 mg, 850 mg-2000 mg, 900 mg-2000 mg, 950 mg-2000 mg, or 1000 mg-2000 mg. In some embodiments, the monthly dose may be an amount of antibody between about 225 mg and about 1000 mg (e.g., a dose of about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 500 mg, about 525 mg, about 550 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, or about 1000 mg). These does may be administered to the patient monthly or quarterly. An exemplary dosing regimen comprises administering an initial antibody dose of about 675 mg subcutaneously, followed by a monthly antibody dose of about 225 mg subcutaneously for about two months, e.g., about three months, four months, five months, six months, or 12 months. One dosing regimen comprises administering an initial antibody dose of about 675 mg intravenously in an infusion over about 60 minutes, followed by doses of about 675 mg administered intravenously in an infusion over about 60 minutes every quarter for one year, two years, three years, four years, or five years. Yet another dosing regimen comprises administering an initial antibody dose of about 900 mg intravenously in an infusion over about 60 minutes, followed by doses of about 900 mg administered intravenously in an infusion over about 60 minutes every quarter for one year, two years, three years, four years, or five years. However, other dosage regimens may be useful, depending on the pattern of pharmacokinetic decay that the practitioner wishes to achieve. In some embodiments, the initial dose and one or more of the additional doses are administered via the same route, e.g., subcutaneously or intravenously. In some embodiments, the one or more additional doses are administered in a different way than the initial dose, e.g., the initial dose may be administered intravenously and the one or more additional doses may be administered subcutaneously.

Alternatively, an antibody described herein may be administered to a subject based on a body weight-based dosing (e.g., an initial candidate dosage can be about 2 mg/kg). In some embodiments, the dose or amount of an antibody (e.g., monoclonal antibody that modulates the CGRP pathway (e.g., an anti-CGRP antagonist antibody or an anti-CGRP receptor antibody)) described herein and administered to a subject may range from about 0.1 to 500, 0.1 to 100, 0.1 to 50, 0.1 to 20, 0.1 to 10, 1 to 10, 1 to 7, 1 to 5 or 0.1 to 3 mg/kg of body weight. In some embodiments, the dose or amount of an antibody described herein and administered to a subject may be, may be at most, may be less than, or may be at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or about 500 mg/kg of body weight. For example, a daily dosage might range from about any of 3 μg/kg to 30 μg/kg to 300 μg/kg to 3 mg/kg, to 30 mg/kg to 100 mg/kg or more. For example, dosage of about 1 mg/kg, about 2.5 mg/kg, about 5 mg/kg, about 10 mg/kg, about 25 mg/kg, and about 30 mg/kg may be used.

The monoclonal antibody that modulates the CGRP pathway can be administered as part of any useful formulation and in any formulation volume. Particularly useful is a formulation comprising the antibody at a concentration of at least about 150 mg/mL (e.g., about 175 mg/mL, about 200 mg/mL, about 225 mg/mL, about 250 mg/mL, about 275 mg/mL, about 300 mg/mL, about 325 mg/mL, about 350 mg/mL, about 375 mg/mL, about 400 mg/mL, about 425 mg/mL, about 450 mg/mL or more).

In some embodiments, a dose (or sub-dose) or amount of an antibody described herein may be formulated in a liquid formulation and administered (e.g., via subcutaneous injection, or via intravenous injection) to a subject. In such cases, the volume of liquid formulation comprising antibody may vary depending upon, for example, the concentration of antibody in the liquid formulation, the desired dose of antibody, and/or the route of administration used. Also useful are formulations wherein the monoclonal antibody can be administered in a volume of less than 2 mL (e.g., about 1.8 mL, about 1.7 mL, about 1.6 mL, about 1.5 mL, about 1.4 ml, about 1.3 mL, about 1.2 mL, about 1.1 mL, about 1.0 ml, about 0.9 mL, about 0.8 mL, about 0.7 mL, about 0.6 mL, about 0.5 mL, or less). Any of the doses provided herein (e.g., about 225 mg, about 675 mg, or about 900 mg) may be administered intravenously or subcutaneously. For example, fremanezumab may be administered at a dose of about 225 mg monthly or quarterly, and be administered subcutaneously.

An antibody formulation may comprise one or more components including the antibody and other species described elsewhere herein. The antibody and other components may be in any suitable amount and/or any suitable concentration for therapeutic efficacy of the antibody, safety and storage. In some embodiments, a liquid formulation comprising an antibody (e.g., monoclonal antibody that modulates the CGRP pathway described herein (e.g., anti-CGRP antagonist antibody or an anti-CGRP receptor antibody) may be prepared for any suitable route of administration with an antibody concentration ranging from about 0.1 mg/mL to about 500 mg/mL, about 0.1 mg/mL to about 375 mg/mL, about 0.1 mg/mL to about 250 mg/mL, about 0.1 to about 175 mg/mL, about 0.1 to 100 mg/mL, about 1 mg/mL to about 500 mg/mL, about 1 mg/mL to about 375 mg/mL, about 1 mg/mL to about 300 mg/mL, about 1 mg/mL to 250 mg/mL, about 1 mg/mL to 200 mg/mL, about 1 mg/mL to 150 mg/mL, about 1 mg/mL to about 100 mg/mL, about 10 mg/mL to 500 mg/mL, about 10 mg/mL to about 375 mg/mL, about 10 mg/mL to 250 mg/mL, about 10 mg/mL to about 150 mg/mL, about 10 mg/mL to 100 mg/mL, about 100 mg/mL to 500 mg/mL, about 100 mg/mL to 450 mg/mL, about 100 mg/mL to 400 mg/mL, about 100 mg/mL to about 350 mg/mL, about 100 mg/mL to about 300 mg/mL, about 100 mg/mL to about 250 mg/mL, 100 mg/mL to 200 mg/mL, or about 100 mg/mL to about 150 mg/mL. In some embodiments, a liquid formulation may comprise an antibody described herein at a concentration of, of at most, of at least, or less than about 0.1, 0.5, 1, 5, 10, 15 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or about 500 mg/mL.

Suitable administration schedules include, but are not limited to, monthly, quarterly, or a single dose. In some embodiments, the monoclonal antibody can be administered monthly. For example, the monoclonal antibody can be administered monthly for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months. In some aspects, the monoclonal antibody can be administered monthly for three or more months.

In some embodiments, the number of doses of antibody administered to a subject over the course of treatment may vary depending upon, for example, achieving reduced incidence of a PIH, e.g., migrainous PIH and non-migrainous PIH, and/or secondary symptom associated with a migrainous PIH in the subject. For example, the number of doses administered over the course of treatment may be, may be at least, or may be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or treatment may be given indefinitely. In some cases, treatment may be acute such that at most 1, 2, 3, 4, 5, or 6 doses are administered to a subject for treatment.

In some embodiments, the frequency at which a dose or amount of an antibody described herein is administered to a subject may vary. In some embodiments, a single dose of antibody may be given to a subject during therapy. In some embodiments, the frequency at which a dose or amount of an antibody is administered to a subject is constant (e.g., administered about once per month or about once per quarter). In some embodiments, the frequency at which a dose or amount of an antibody is administered to a subject is about every quarter for about one year, two years, three years, four years, or five years. In some embodiments, the frequency at which a dose or amount of an antibody described herein is administered to a subject is variable (e.g., an initial dose followed by a dose at once per month, followed by additional doses at about three months and about seven months). In some embodiments, the frequency at which an antibody is administered to a subject is, is at least, is less than, or is at most about one, two, three, four, five, or six time(s) per day. In some embodiments, the frequency at which an antibody described herein is administered to a subject is, is at least, is less than, or is at most about one, two, three, four, five, or six dose(s) per day. In some embodiments, the frequency at which a dose or amount of an antibody described herein is administered to a subject is, is at least, is less than, or is at most one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty time(s) per every one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-nine, forty, forty-one, forty-two, forty-three, forty-four, forty-five, forty-six, forty-seven, forty-eight, forty-nine, fifty, fifty-five, sixty, sixty-five, seventy, seventy-five, eighty, eighty-five, ninety, ninety-five, one-hundred, one-hundred twenty-five, one-hundred fifty, one-hundred eighty, or two-hundred day(s).

In some embodiments, the frequency at which a dose or amount of an antibody (e.g., monoclonal antibody that modulates the CGRP pathway described herein (e.g., an anti-CGRP antagonist antibody or an anti-CGRP receptor antibody) is administered to a subject is at least, is less than, or is at most one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty time(s) per every one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-nine, forty, forty-one, forty-two, forty-three, forty-four, forty-five, forty-six, forty-seven, forty-eight, forty-nine, fifty, fifty-five, sixty, sixty-five, seventy, seventy-five, eighty, eighty-five, ninety, ninety-five, or one-hundred week(s). In some embodiments, the frequency at which an antibody described herein is administered to a subject is less than one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen dose(s) per week.

In some embodiments, the frequency at which a dose or amount of an antibody described herein is administered to a subject is, is at least, is less than, or is at most about one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty time(s) per every month, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, every twelve months, every thirteen months, every fourteen months, every fifteen months, every sixteen months, every seventeen months, or every eighteen month(s). In some embodiments, the frequency at which a dose or amount of an antibody described herein is administered to a subject is about one time per every one month. In some embodiments, the frequency at which a dose or amount of an antibody described herein is administered to a subject is about one time per every three months. In some embodiments, the frequency at which an antibody described herein is administered to a subject is less than about one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen dose(s) per month. In some embodiments, a dose or amount of an antibody described herein may be administered to a subject one time, two times, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more per month.

The appropriate dosage of an antibody described herein may depend on the antibody (or compositions thereof) employed, the type and severity of the secondary symptom associated with a migrainous PIH, the type and severity of the PIH or other condition to be treated, whether the agent is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the agent, and the discretion of the attending physician. Typically, the clinician will administer an antibody, until a dosage is reached that achieves the desired result. Dose and/or frequency can vary over course of treatment. In one embodiment, dosages for an antibody described herein may be determined empirically in individuals who have been given one or more administration(s) of the antibody. Individuals are given incremental dosages of an antibody. To assess efficacy of an antibody, an indicator of the disease can be followed.

Empirical considerations, such as the half-life, generally will contribute to the determination of the dosage. For example, antibodies that are compatible with the human immune system, such as humanized antibodies or fully human antibodies, may be used to prolong half-life of the antibody and to prevent the antibody being attacked by the host's immune system. Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or amelioration and/or delay of PIH or other condition. Alternatively, sustained continuous release formulations of antibodies may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

With respect to all methods described herein, references to antibodies (e.g., monoclonal antibodies that modulate the CGRP pathway (e.g., anti-CGRP antagonist antibodies or anti-CGRP receptor antibodies) also include compositions comprising one or more of these agents. Accordingly, such a composition may be used according to a method referring to an antibody described herein. These compositions may further comprise suitable excipients, such as pharmaceutically acceptable excipients as described elsewhere herein. The present invention can be used alone or in combination with other conventional methods of treatment.

An antibody described herein (e.g., an anti-CGRP antagonist antibody or an anti-CGRP receptor antibody) can be administered to an individual or subject in any therapeutic dose, via any suitable route and in any suitable formulation. It should be apparent to a person skilled in the art that the examples described herein are not intended to be limiting but to be illustrative of the techniques available. Accordingly, in some embodiments, an antibody described herein can be administered to a subject in accord with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, e.g., about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 180 minutes, or about 240 minutes. The antibody described herein can also be administered to the subject by subcutaneous, intramuscular, intraperitoneal, intracerebrospinal, intra-articular, sublingually, intra-arterial, intrasynovial, via insufflation, intrathecal, oral, inhalation, intranasal (e.g., with or without inhalation), buccal, rectal, transdermal, intracardiac, intraosseous, intradermal, transmucosal, vaginal, intravitreal, peri-articular, local, epicutaneous, or topical routes. Administration can be systemic, e.g., intravenous administration, or localized. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers are useful for administration. Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution. Alternatively, an antibody described herein can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.

In some embodiments, an antibody described herein can be administered via site-specific or targeted local delivery techniques. Examples of site-specific or targeted local delivery techniques include various implantable depot sources of the antibody or local delivery catheters, such as infusion catheters, an indwelling catheter, or a needle catheter, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site specific carriers, direct injection, or direct application. See e.g., PCT Publication No. WO 00/53211 and U.S. Pat. No. 5,981,568, which are hereby incorporated by reference in their entireties.

Various formulations of an antibody described herein may be used for administration. In some embodiments, an antibody may be administered neat. In some embodiments, antibody and a pharmaceutically acceptable excipient may be in various formulations. Pharmaceutically acceptable excipients are known in the art, and are relatively inert substances that facilitate administration of a pharmacologically effective substance. For example, an excipient can give form or consistency, or act as a diluent. Suitable excipients include but are not limited to stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers. Excipients as well as formulations for parenteral and nonparenteral drug delivery are set forth in Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

Skilled practitioners will appreciate that the antibody(ies) can be administered to the patient using any method known in the art. For example, the antibody(ies) can be administered to the patient using a pre-filled syringe, a pre-filled syringe with a needle safety device, an injection pen, an auto-injector, or any combination thereof.

Administration of an antibody (e.g., monoclonal antibody that modulates the CGRP pathway (e.g., a monoclonal anti-CGRP antagonist antibody or a monoclonal anti-CGRP receptor antibody) in accordance with the methods of the present invention can be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of an antibody may be essentially continuous over a preselected period of time or may be in a series of spaced dose, e.g., either before, during, or after developing a seizure; before; during; before and after; during and after; before and during; or before, during, and after developing a seizure. Administration can be before, during, and/or after any event likely to give rise to a seizure, e.g., at least about five minutes prior to a seizure, e.g., at least about ten minutes, at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about one hour, at least about two hours, at least about three hours, at least about four hours, at least about five hours, at least about six hours, at least about seven hours, at least about eight hours, at least about nine hours, at least about ten hours, at least about 12 hours, at least about 18 hours, at least about 24 hours, at least about 48 hours, or at least about 72 hours prior to a seizure.

In some embodiments, more than one antibody may be present. At least one, at least two, at least three, at least four, at least five different, or more antibodies can be present. Generally, those antibodies may have complementary activities that do not adversely affect each other. An antibody (e.g., a monoclonal antibody that modulates the CGRP pathway (e.g., an anti-CGRP antagonist antibody or an anti-CGRP receptor antibody)) described herein can also be used in conjunction with other CGRP antagonists or CGRP receptor antagonists. For example, one or more of the following CGRP antagonists may be used: an anti-sense molecule directed to a CGRP (including an anti-sense molecule directed to a nucleic acid encoding CGRP), a CGRP inhibitory compound, a CGRP structural analog, a dominant-negative mutation of a CGRP receptor that binds a CGRP, and an anti-CGRP receptor antibody. An antibody can also be used in conjunction with other agents that serve to enhance and/or complement the effectiveness of the agents.

Diagnosis or assessment of PIH is well-established in the art. Assessment may be performed based on subjective measures, such as patient characterization of symptoms. In some embodiments, assessment of PIH may be via headache or migraine hours, as described elsewhere herein. For example, assessment of PIH may be in terms of daily headache or migraine hours, weekly headache or migraine hours, and/or monthly headache or migraine hours. In some cases, headache or migraine hours may be as reported by the subject.

Treatment efficacy can be assessed by methods well-known in the art. For example, pain relief may be assessed. Accordingly, in some embodiments, pain relief is subjectively observed after 1, 2, or a few hours after administering an anti-CGRP antibody.

In some embodiments, a method for preventing, treating, or reducing incidence of PIH in a subject as described herein may reduce incidence of PIH after a single administration of an antibody (e.g., monoclonal antibody that modulates the CGRP pathway (e.g., a monoclonal anti-CGRP antagonist antibody or an anti-CGRP receptor antibody) described herein for an extended period of time. For example, incidence of PIH may be reduced for at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more days after a single administration.

PIHs can be short, lasting only several hours, or they can progress to a typical migraine attack. For short PIHs, the relevant endpoint is a reduction in headache hours after a seizure. For longer PIHs that progress to a typical migraine attack, a reduction in headache hours, headache days, migraine hours, migraine days (per day or per month) can be relevant.

The preventing, treating, or reducing can comprise reducing the number of headache hours of any severity, reducing the number of migraine hours of any severity, reducing the number of monthly headache days of any severity, reducing the number of monthly migraine days of any severity, reducing the use of any acute headache medications, reducing a 6-item Headache Impact Test (HIT-6) disability score, improving 12-Item Short Form Health Survey (SF-12) score (Ware et al., Med Care 4:220-233, 1996), reducing Patient Global Impression of Change (PGIC) score (Hurst et al., J. Manipulative. Physiol. Ther. 27:26-35, 2004), improving Sport ConCuSSion ASSeSment tool 3 (SCAT-3) score (McCrory et al. British Journal of Sports Medicine 47:263-266, 2013), or any combination thereof.

In some embodiments, a method for preventing, treating, or reducing incidence of PIH in a subject as described herein may reduce the number of headache or migraine hours experienced by a subject from a pre-administration level after administration of one or more doses of an antibody (e.g., a monoclonal antibody that modulates the CGRP pathway (e.g., a monoclonal anti-CGRP antagonist antibody or a monoclonal anti-CGRP receptor antibody) described herein to the subject. For example, daily headache or migraine hours experienced by the subject after administering one or more doses of an antibody described herein to the subject may be reduced by 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 headache or migraine hours from a pre-administration level in the subject. In some cases, daily headache or migraine hours experienced by the subject after administering one or more doses of an antibody described herein to the subject may be reduced by 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more relative to a pre-administration level in the subject. In another example, weekly headache or migraine hours experienced by the subject after administering one or more doses of an antibody described herein to the subject may be reduced by 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more headache or migraine hours from a pre-administration level in the subject. In some cases, weekly headache or migraine hours experienced by the subject after administering one or more doses of an antibody described herein to the subject may be reduced by 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more relative to a pre-administration level in the subject. In another example, monthly headache or migraine hours experienced by the subject after administering one or more doses of an antibody described herein to the subject may be reduced by 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or more headache or migraine hours from a pre-administration level. In some cases, weekly headache or migraine hours experienced by the subject after administering one or more doses of an antibody described herein to the subject may be reduced by 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more relative to a pre-administration level in the subject. In some embodiments, the number of monthly headache or migraine days can be reduced for at least seven days after a single administration.

In some embodiments, a method for preventing, treating, or reducing incidence of PIH in a subject as described herein may reduce the number of headache or migraine days experienced by a subject from a pre-administration level after administration of one or more doses of an antibody (e.g., a monoclonal antibody that modulates the CGRP pathway (e.g., a monoclonal anti-CGRP antagonist antibody or a monoclonal anti-CGRP receptor antibody) described herein to the subject. For example, weekly headache or migraine days experienced by the subject after administering one or more doses of an antibody to the subject may be reduced by 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7 headache or migraine days from a pre-administration level in the subject. In some cases, weekly headache or migraine days experienced by the subject after administering one or more doses of an antibody described herein to the subject may be reduced by 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more relative to a pre-administration level in the subject. In another example, monthly headache or migraine days experienced by the subject after administering one or more doses of an antibody described herein to the subject may be reduced by 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more headache or migraine days from a pre-administration level.

B. Anti-CGRP Antibodies

In some embodiments, the methods of the invention use an antibody, which can be an anti-CGRP antagonist antibody. An anti-CGRP antagonist antibody can refer to any antibody molecule that blocks, suppresses or reduces (including significantly) CGRP biological activity, including downstream pathways mediated by CGRP signaling, such as receptor binding and/or elicitation of a cellular response to CGRP.

An anti-CGRP antagonist antibody can exhibit any one or more of the following characteristics: (a) bind to CGRP; (b) block CGRP from binding to its receptor(s); (c) block or decrease CGRP receptor activation (including, but not limited to, cAMP activation); (d) inhibit CGRP biological activity or downstream pathways mediated by CGRP signaling function; (e) prevent, ameliorate, reduce severity of, or treat any aspect of PIH; (f) increase clearance of CGRP; and (g) inhibit (reduce) CGRP synthesis, production or release. Anti-CGRP antagonist antibodies are known in the art. See e.g., Tan et al., Clin. Sci. (Lond). 89:565-73, 1995; Sigma (Missouri, US), product number C7113 (clone #4901); Plourde et al., Peptides 14:1225-1229, 1993.

In some embodiments, the antibody reacts with CGRP in a manner that inhibits CGRP, and/or the CGRP pathway, including downstream pathways mediated by the CGRP signaling function. In some embodiments, the anti-CGRP antagonist antibody recognizes human CGRP. In some embodiments, the anti-CGRP antagonist antibody binds to both human α-CGRP and β-CGRP. In some embodiments, the anti-CGRP antagonist antibody binds human and rat CGRP. In some embodiments, the anti-CGRP antagonist antibody binds the C-terminal fragment having amino acids 25-37 of CGRP. In some embodiments, the anti-CGRP antagonist antibody binds a C-terminal epitope within amino acids 25-37 of CGRP.

The anti-CGRP antibodies useful in the present invention can encompass monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab′, F(ab′)2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion (e.g., a domain antibody), humanized antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibodies may be murine, rat, human, or any other origin (including chimeric or humanized antibodies).

In some embodiments, the anti-CGRP antagonist antibody is a monoclonal antibody. In some embodiments, the anti-CGRP antagonist antibody is humanized. In some embodiments, the antibody is human. In some embodiments, the anti-CGRP antagonist antibody is antibody G1 (as described herein). In some embodiments, the anti-CGRP antagonist antibody comprises one or more CDR(s) (such as one, two, three, four, five, or, in some embodiments, all six CDRs) of antibody G1 or variants of G1 shown in Table 5. In still other embodiments, the anti-CGRP antagonist antibody comprises the amino acid sequence of the heavy chain variable region shown in FIG. 5 (SEQ ID NO:1) and the amino acid sequence of the light chain variable region shown in FIG. 5 (SEQ ID NO:2). In still other embodiments, the anti-CGRP antagonist antibody comprises a heavy chain full length amino acid sequence shown in SEQ ID NO:11, and a light chain full length amino acid sequence shown if SEQ ID NO:12.

In some embodiments, the antibody comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR) selected from the groups consisting of: (a) LCVR17 (SEQ ID NO:58) and HCVR22 (SEQ ID NO:59); (b) LCVR18 (SEQ ID NO:60) and HCVR23 (SEQ ID NO:61); (c) LCVR19 (SEQ ID NO:62) and HCVR24 (SEQ ID NO:63); (d) LCVR20 (SEQ ID NO:64) and HCVR25 (SEQ ID NO:65); (e) LCVR21 (SEQ ID NO:66) and HCVR26 (SEQ ID NO:67); (f) LCVR27 (SEQ ID NO:68) and HCVR28 (SEQ ID NO:69); (g) LCVR29 (SEQ ID NO:70) and HCVR30 (SEQ ID NO:71); (h) LCVR31 (SEQ ID NO:72) and HCVR32 (SEQ ID NO:73); (i) LCVR33 (SEQ ID NO:74) and HCVR34 (SEQ ID NO:75); (j) LCVR35 (SEQ ID NO:76) and HCVR36 (SEQ ID NO:77); and (k) LCVR37 (SEQ ID NO:78) and HCVR38 (SEQ ID NO:79). Sequences of these regions are provided herein. Other examples of anti-CGRP antibodies are described in U.S Patent Publication Nos. US 2011/0305711 (SEQ ID NOs:5, 6, 7, 12, 16, 19, 24, 29, 34, and 39), US 2012/0294802, US 2012/0294797 (SEQ ID NOs:51-60), which are hereby incorporated by reference in their entireties. For example, antibodies with any of the following sequences may be used.

Ab6 Variable region Light chain (humanized) protein sequence (US20120294797) (SEQ ID NO: 80) QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKVPKQLIYDASTLASGVPSRFSGSGSGTDFTLTISSLQPED VATYYCLGSYDCTNGDCFVFGGGTKVEIKR Ab6 Light chain (humanized) Full length protein sequence (US20120294797) (SEQ ID NO: 81) QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKVPKQLIYDASTLASGVPSRFSGSGSGTDFTLTISSLQPED VATYYCLGSYDCTNGDCFVFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Ab6 Variable region heavy chain (humanized) protein sequence (US20120294797) (SEQ ID NO: 82) EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGINGATYYASWAKGRFTISRDNSKTTVYLQMN SLRAEDTAVYFCARGDIWGQGTLVTVSS Ab6 Heavy chain (humanized) Full length protein sequence-yeast produced (US20120294797) (SEQ ID NO: 83) EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGINGATYYASWAKGRFTISRDNSKTTVYLQMN SLRAEDTAVYFCARGDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK Ab6 Variable region Light chain (humanized) protein sequence CDR1 (US20120294797) (SEQ ID NO: 84) QASQSVYHNTYLA Ab6 Variable region Light chain (humanized) protein sequence CDR2 (US20120294797) (SEQ ID NO: 85) DASTLAS Ab6 Variable region Light chain (humanized) protein sequence CDR3 (US20120294797) (SEQ ID NO: 86) LGSYDCTNGDCFV Ab6 Variable region heavy chain (humanized) protein sequence CDR1 (US20120294797) (SEQ ID NO: 87) GYYMN Ab6 Variable region heavy chain (humanized) protein sequence CDR2 (US20120294797) (SEQ ID NO: 88) IGINGATYYASWAKG Ab6 Variable region heavy chain (humanized) protein sequence CDR3 (US20120294797) (SEQ ID NO: 89) GDI Light chain variable region protein sequence CDR3 (US20110305711) (SEQ ID NO: 90) QQGDALPPT Light chain variable region protein sequence CDR1 (US20110305711) (SEQ ID NO: 91) RASKDISKYL Light chain variable region protein sequence CDR2 (US20110305711) (SEQ ID NO: 92) YTSGYHS Heavy chain variable region protein sequence CDR1 (US20110305711) (SEQ ID NO: 93) GYTFGNYWMQ Heavy chain variable region protein sequence CDR2 (US20110305711) (SEQ ID NO: 94) AIYEGTGKTVYIQKFAD Heavy chain variable region protein sequence CDR3 (US20110305711) (SEQ ID NO: 95) LSDYVSGFGY Light chain variable region protein sequence (US20110305711) (SEQ ID NO: 96) DIQMTQSPSSLSASVGDRVTITCRASKDISKYLNWYQQKPGKAPKLLIYYTSGYHSGVPSRFSGSGSGTDFTLTISSLQPEDF ATYYCQQGDALPPTFGGGTKVEIK Heavy chain variable region protein sequence (US20110305711) (SEQ ID NO: 97) QVQLVQSGAEVKKPGSSVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGTGKTVYIQKFADRVTITADKSTSTAYMEL SSLRSEDTAVYYCARLSDYVSGFGYWGQGTTVTVSS Light chain protein sequence (US20110305711) (SEQ ID NO: 98) DIQMTQSPSSLSASVGDRVTITCRASKDISKYLNWYQQKPGKAPKLLIYYTSGYHSGVPSRFSGSGSGTDFTLTISSLQPEDF ATYYCQQGDALPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Heavy chain protein sequence (US20110305711) (SEQ ID NO: 99) QVQLVQSGAEVKKPGSSVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGTGKTVYIQKFADRVTITADKSTSTAYMEL SSLRSEDTAVYYCARLSDYVSGFGYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR WQEGNVFSCSVMHEALHNHYTQKSLSLSLG

In some embodiments, the antibody comprises a modified constant region, such as a constant region that is immunologically inert described herein.

The binding affinity (K_(D)) of an anti-CGRP antagonist antibody to CGRP (such as human α-CGRP) can be about 0.02 to about 200 nM. In some embodiments, the binding affinity is any of about 200 nM, about 100 nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, about 60 pM, about 50 pM, about 20 pM, about 15 pM, about 10 pM, about 5 pM, or about 2 pM. In some embodiments, the binding affinity is less than any of about 250 nM, about 200 nM, about 100 nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, or about 50 pM.

In some embodiments, an anti-CGRP receptor antibody can be used in any of the methods described herein. For example, anti-CGRP receptor antibodies, as described in U.S. Patent Publication No. US 2010/0172895 and U.S. Pat. No. 9,102,731, which are hereby incorporated by reference in their entireties, may be used. Therefore, antibodies with any of the following sequences may be used.

Light chain variable region protein sequence CDR1 (U.S. Pat. No. 9,102,731) (SEQ ID NO: 100) SGSSSNIGNNYVS Light chain variable region protein sequence CDR2 (U.S. Pat. No. 9,102,731) (SEQ ID NO: 101) DNNKRPS Light chain variable region protein sequence CDR3 (U.S. Pat. No. 9,102,731) (SEQ ID NO: 102) GTWDSRLSAVV Heavy chain variable region protein sequence CDR1 (U.S. Pat. No. 9,102,731) (SEQ ID NO: 103) SFGMH Heavy chain variable region protein sequence CDR2 (U.S. Pat. No. 9,102,731) (SEQ ID NO: 104) VISFDGSIKYSVDSVKG Heavy chain variable region protein sequence CDR3 (U.S. Pat. No. 9,102,731) (SEQ ID NO: 105) DRLNYYDSSGYYHYKYYGMAV Light chain variable region protein sequence (U.S. Pat. No. 9,102,731) (SEQ ID NO: 106) QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIY DNNKRPSGIPDRFSGSKSGTSTTLGITGLQTGDEADYYCGTWDSRLSAVV FGGGTKLTVL Heavy chain variable region protein sequence (U.S. Pat. No. 9,102,731) (SEQ ID NO: 107) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAV ISFDGSIKYSVDSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCARDR LNYYDSSGYYHYKYYGMAVWGQGTTVTVSS Light chain protein sequence (U.S. Pat. No. 9,102,731) (SEQ ID NO: 108) MDMRVPAQLLGLLLLWLRGARCQSVLTQPPSVSAAPGQKVTISCSGSSSN IGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSTTLGITG LQTGDEADYYCGTWDSRLSAVVFGGGTKLTVLGQPKANPTVTLFPPSSEE LQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAA SSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Heavy chain protein sequence (U.S. Pat. No. 9,102,731) (SEQ ID NO: 109) MDMRVPAQLLGLLLLWLRGARCQVQLVESGGGVVQPGRSLRLSCAASGFT FSSFGMHWVRQAPGKGLEWVAVISFDGSIKYSVDSVKGRFTISRDNSKNT LFLQMNSLRAEDTAVYYCARDRLNYYDSSGYYHYKYYGMAVWGQGTTVTV SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEY KCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK

C. Antibody G1 and Related Antibodies, Polypeptides, Polynucleotides, Vectors and Host Cells

Provided herein are methods of preventing a post-ictal headache in a subject including using compositions (e.g., pharmaceutical compositions) comprising antibody G1 and its variants shown in Table 5 or polypeptide derived from antibody G1 and its variants shown in Table 5; and polynucleotides comprising sequences encoding G1 and its variants or the polypeptide. In some embodiments, the compositions used in the methods provided herein comprise one or more antibodies or polypeptides (which may or may not be an antibody) that bind to CGRP, and/or one or more polynucleotides comprising sequences encoding one or more antibodies or polypeptides that bind to CGRP. These compositions may further comprise suitable excipients, such as pharmaceutically acceptable excipients including buffers, which are well known in the art.

Anti-CGRP antagonist antibodies and polypeptides useful in the methods described herein may be characterized by any (one or more) of the following characteristics: (a) bind to CGRP; (b) block CGRP from binding to its receptor(s); (c) block or decrease CGRP receptor activation (including cAMP activation); (d) inhibit CGRP biological activity or downstream pathways mediated by CGRP signaling function; (e) prevent, ameliorate, reduce severity of, or treat any aspect of PIH; (f) increase clearance of CGRP; and (g) inhibit (reduce) CGRP synthesis, production or release.

Useful in the methods described herein are any of the following, or compositions (including pharmaceutical compositions) comprising any of the following: (a) antibody G1 or its variants shown in Table 5 (b) a fragment or a region of antibody G1 or its variants shown in Table 5; (c) a light chain of antibody G1 or its variants shown in Table 5; (d) a heavy chain of antibody G1 or its variants shown in Table 5; (e) one or more variable region(s) from a light chain and/or a heavy chain of antibody G1 or its variants shown in Table 5; (f) one or more CDR(s) (one, two, three, four, five or six CDRs) of antibody G1 or its variants shown in Table 5; (g) CDR H3 from the heavy chain of antibody G1; (h) CDR L3 from the light chain of antibody G1 or its variants shown in Table 5; (i) three CDRs from the light chain of antibody G1 or its variants shown in Table 5; (j) three CDRs from the heavy chain of antibody G1 or its variants shown in Table 5; (k) three CDRs from the light chain and three CDRs from the heavy chain, of antibody G1 or its variants shown in Table 5; and (l) an antibody comprising any one of (b) through (k). In some embodiments, the methods include using polypeptides comprising any one or more of the above.

The CDR portions of antibody G1 (including Chothia and Kabat CDRs) are diagrammatically depicted in FIG. 5. Determination of CDR regions is well within the skill of the art. Skilled practitioners will appreciate that CDRs can be a combination of the Kabat and Chothia CDR (also termed “combined CDRs” or “extended CDRs”). In some instances, the CDRs are the Kabat CDRs and in others the CDRs are the Chothia CDRs. In other words, in some instances where more than one CDR are useful, the CDRs may be any of Kabat, Chothia, combination CDRs, or combinations thereof.

Methods described herein can employ a polypeptide (which may or may not be an antibody) which comprises at least one CDR, at least two, at least three, or at least four, at least five, or all six CDRs that are substantially identical to at least one CDR, at least two, at least three, at least four, at least five or all six CDRs of G1 or its variants shown in Table 5. The methods include using antibodies which have at least two, three, four, five, or six CDR(s) that are substantially identical to at least two, three, four, five or six CDRs of G1 or derived from G1. In some instances, the at least one, two, three, four, five, or six CDR(s) are at least about 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, or 99% identical to at least one, two, three, four, five or six CDRs of G1 or its variants shown in Table 5.

Methods provided herein can utilize a polypeptide (which may or may not be an antibody) which comprises an amino acid sequence of G1 or its variants shown in Table 5 that has any of the following: at least 5 contiguous amino acids, at least 8 contiguous amino acids, at least about 10 contiguous amino acids, at least about 15 contiguous amino acids, at least about 20 contiguous amino acids, at least about 25 contiguous amino acids, at least about 30 contiguous amino acids of a sequence of G1 or its variants shown in Table 5, wherein at least 3 of the amino acids are from a variable region of G1 (FIG. 5) or its variants shown in Table 5. For example, the variable region can be from a light chain or from a heavy chain of G1. An exemplary polypeptide has contiguous amino acid (lengths described above) from both the heavy and light chain variable regions of G1. In another embodiment, the 5 (or more) contiguous amino acids are from a complementarity determining region (CDR) of G1 shown in FIG. 5. In some embodiments, the contiguous amino acids are from a variable region of G1.

The binding affinity (K_(D)) of an anti-CGRP antagonist antibody and polypeptide to CGRP, as used in the methods provided herein, (such as human α-CGRP) can be about 0.06 to about 200 nM. For example, the binding affinity can be any of about 200 nM, 100 nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, about 60 pM, about 50 pM, about 20 pM, about 15 pM, about 10 pM, about 5 pM, or about 2 pM. In other examples, the binding affinity is less than any of about 250 nM, about 200 nM, about 100 nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, or about 50 pM.

The methods provided herein may use single chain variable region fragments (“scFv”) of antibodies described herein, such as G1. Single chain variable region fragments are made by linking light and/or heavy chain variable regions by using a short linking peptide. Bird et al. (1988) Science 242:423-426.

Humanized antibody comprising one or more CDRs of antibody G1 or its variants shown in Table 5, or one or more CDRs derived from antibody G1 or its variants shown in Table 5 can be made using any methods known in the art.

In some instances, methods described herein can employ using antibody G1 comprising modifications such as those shown in Table 5, including functionally equivalent antibodies which do not significantly affect their properties and variants which have enhanced or decreased activity and/or affinity. For example, the amino acid sequence of antibody G1 or its variants shown in Table 5 may be mutated to obtain an antibody with the desired binding affinity to CGRP. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, one or more deletions or additions of amino acids which do not significantly deleteriously change the functional activity, or use of chemical analogs.

Modifications also include glycosylated and nonglycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation, and phosphorylation. Techniques to achieve this type of modification are well known in the art.

Compositions (such as a pharmaceutical compositions) comprising any of the polynucleotides encoding polypeptides described herein can be used in the presently described methods. In some instances, the composition comprises an expression vector comprising a polynucleotide encoding the G1 antibody and/or any of the antibodies or polypeptides described herein. For example, the composition can include either or both of the polynucleotides shown in SEQ ID NO:9 and SEQ ID NO:10. Useful expression vectors, and methods of administering polynucleotide compositions are known in the art and further described herein.

D. Compositions

In some embodiments, compositions used in a method described herein comprise an effective amount of an antibody that modulates the CGRP pathway (e.g., an anti-CGRP antagonist antibody or an anti-CGRP receptor antibody) or an antibody-derived polypeptide described herein. A composition (e.g., a medicament or therapeutic formulation) can further comprise pharmaceutically acceptable carriers, excipients, or stabilizers (Remington: The Science and practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed.

An antibody (e.g., an anti-CGRP antagonist antibody or an anti-CGRP receptor antibody) and compositions thereof provided herein can also be used in conjunction with other agents that serve to enhance and/or complement the effectiveness of the antibody.

E. Kits

Also provided herein are kits for use in the instant methods. Kits can include one or more containers comprising an antibody described herein (e.g., an anti-CGRP antagonist antibody (such as a humanized antibody)) or polypeptide described herein and instructions for use in accordance with any of the methods described herein. Generally, these instructions comprise a description of administration of the antibody to treat, ameliorate, reduce severity of, or prevent PIH according to any of the methods described herein. For example, the kit may comprise a description of how to select an individual suitable for treatment based on identifying whether that individual has a seizure or whether the individual is at risk of having PIH. In still other embodiments, the instructions include a description of how to administer an antibody (e.g., anti-CGRP antagonist antibody) to an individual at risk of having PIH.

Accordingly, a kit can include, e.g., a pre-filled syringe, a pre-filled syringe with a needle safety device, an injection pen, or an auto-injector comprising a dose of a monoclonal antibody that modulates the CGRP pathway; and instructions for use in accordance with any of the methods described herein.

The kit may further comprise instructions relating to the use of the antibody (e.g., for treating, ameliorating, reducing severity of, and/or preventing PIH) including information as to dosage, dosing schedule, and route of administration for the intended treatment. The kit may be include unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

In a kit provided herein, a monoclonal antibody provided in the kit can include a CDR H1 as set forth in SEQ ID NO:3; a CDR H2 as set forth in SEQ ID NO:4; a CDR H3 as set forth in SEQ ID NO:5; a CDR L1 as set forth in SEQ ID NO:6; a CDR L2 as set forth in SEQ ID NO:7; and a CDR L3 as set forth in SEQ ID NO:8. In some embodiments, a monoclonal antibody provided in a kit comprises a heavy chain variable region comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:1, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, a monoclonal antibody provided in a kit comprises a heavy chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:11, and a light chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:12.

Alternatively or in addition, a monoclonal antibody provided in a kit can include a CDR H1 as set forth in SEQ ID NO:87; a CDR H2 as set forth in SEQ ID NO:88; a CDR H3 as set forth in SEQ ID NO:89; a CDR L1 as set forth in SEQ ID NO:84; a CDR L2 as set forth in SEQ ID NO:85; and a CDR L3 as set forth in SEQ ID NO:86. In some embodiments, a monoclonal antibody provided in a kit comprises a heavy chain variable region comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:82, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:80. In some embodiments, a monoclonal antibody provided in a kit comprises a heavy chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:83, and a light chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:81.

Alternatively or in addition, a monoclonal antibody provided in a kit can include a CDR H1 as set forth in SEQ ID NO:93; a CDR H2 as set forth in SEQ ID NO:94; a CDR H3 as set forth in SEQ ID NO:95; a CDR L1 as set forth in SEQ ID NO:91; a CDR L2 as set forth in SEQ ID NO:92; and a CDR L3 as set forth in SEQ ID NO:90. In some embodiments, a monoclonal antibody provided in a kit comprises a heavy chain variable region comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:97, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:96. In some embodiments, a monoclonal antibody provided in a kit comprises a heavy chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:99, and a light chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:98.

Alternatively or in addition, a monoclonal antibody provided in a kit can include a CDR H1 as set forth in SEQ ID NO:103; a CDR H2 as set forth in SEQ ID NO:104; a CDR H3 as set forth in SEQ ID NO:105; a CDR L1 as set forth in SEQ ID NO:100; a CDR L2 as set forth in SEQ ID NO:101; and a CDR L3 as set forth in SEQ ID NO:102. In some embodiments, a monoclonal antibody provided in a kit comprises a heavy chain variable region comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:107, and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:106. In some embodiments, a monoclonal antibody provided in a kit comprises a heavy chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:109, and a light chain comprising or consisting of the amino acid sequence as set forth in SEQ ID NO:108.

A monoclonal antibody provided in a kit can be fremanezumab, eptinezumab, galcanezumab, erenumab, or any bioequivalent thereof. Skilled practitioners will appreciate that a kit can include a combination of any of the foregoing antibodies

The kits can be provided in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. A kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody that modulates the CGRP receptor pathway (e.g., an anti-CGRP antagonist antibody or an anti-CGRP receptor antibody). The container may further comprise a second pharmaceutically active agent.

Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container.

The following Examples are provided to illustrate but not limit the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent or patent application were specifically and individually indicated to be so incorporated by reference.

EXAMPLES Example 1: Generation and Characterization of Monoclonal Antibodies Directed Against CGRP

Generation of anti-CGRP antibodies. To generate anti-CGRP antibodies that have cross-species reactivity for rat and human CGRP, mice were immunized with 25-100 μg of human α-CGRP or β-CGRP conjugated to KLH in adjuvant (50 μl per footpad, 100 μl total per mouse) at various intervals. Immunization was generally performed as described in Geerligs H J et al., 1989, J. Immunol. Methods 124:95-102; Kenney J S et al., 1989, J. Immunol. Methods 121:157-166; and Wicher K et al., 1989, Int. Arch. Allergy Appl. Immunol. 89:128-135. Mice were first immunized with 50 μg of human α-CGRP or β-CGRP conjugated to KLH in CFA (complete Freund's adjuvant). After 21 days, mice were secondly immunized with 25 μg of human β-CGRP (for mice first immunized with human α-CGRP) or α-CGRP (for mice first immunized with human β-CGRP) conjugated to KLH in IFA (incomplete Freund's adjuvant). Twenty-three days later after the second immunization, third immunization was performed with 25 μg of rat α-CGRP conjugated to KLH in IFA. Ten days later, antibody titers were tested using ELISA. Forth immunization was performed with 25 μg of the peptide (rat α-CGRP-KLH) in IFA 34 days after the third immunization. Final booster was performed with 100 μg soluble peptide (rat α-CGRP) 32 days after the forth immunization.

Splenocytes were obtained from the immunized mouse and fused with NSO myeloma cells at a ratio of 10:1, with polyethylene glycol 1500. The hybrids were plated out into 96-well plates in DMEM containing 20% horse serum and 2-oxaloacetate/pyruvate/insulin (Sigma), and hypoxanthine/aminopterin/thymidine selection was begun. On day 8, 100 μl of DMEM containing 20% horse serum was added to all the wells. Supernatants of the hybrids were screened by using antibody capture immunoassay. Determination of antibody class was done with class-specific second antibodies.

A panel of monoclonal antibody-producing cell lines was selected based on their binding to human and rat CGRP for further characterization. These antibodies and characteristics are shown below in Tables 2 and 3.

Purification and Fab fragment preparation. Monoclonal antibodies selected for further characterization were purified from supernatants of hybridoma cultures using protein A affinity chromatography. The supernatants were equilibrated to pH 8. The supernatants were then loaded to the protein A column MabSelect (Amersham Biosciences #17-5199-02) equilibrated with PBS to pH 8. The column was washed with 5 column volumes of PBS, pH 8. The antibodies were eluted with 50 mM citrate-phosphate buffer, pH 3. The eluted antibodies were neutralized with 1 M Phosphate Buffer, pH 8. The purified antibodies were dialyzed with PBS, pH 7.4. The antibody concentrations were determined by SDS-PAGE, using a murine monoclonal antibody standard curve.

Fabs were prepared by papain proteolysis of the full antibodies using Immunopure Fab kit (Pierce #44885) and purified by flow through protein A chromatography following manufacturer instructions. Concentrations were determined by ELISA and/or SDS-PAGE electrophoresis using a standard Fab of known concentration (determined by amino acid analysis), and by A280 using 1OD=0.6 mg/ml (or theoretical equivalent based on the amino acid sequence).

Affinity determination of the Fabs. Affinities of the anti-CGRP monoclonal antibodies were determined at either 25° C. or 37° C. using the BIACORE3000™ surface plasmon resonance (SPR) system (Biacore, INC, Piscataway N.J.) with the manufacture's own running buffer, HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% v/v polysorbate P20). Affinity was determined by capturing N-terminally biotinylated CGRP peptides (custom ordered from GenScript Corporation, New Jersey or Global Peptide Services, Colorado) via pre-immobilized streptavidin on SA chip and measuring binding kinetics of antibody Fab titrated across the CGRP surface. Biotinylated CGRP was diluted into HBS-EP and injected over the chip at a concentration of less than 0.001 mg/ml. Using variable flow time across the individual chip channels, two ranges of antigen density were achieved: <50 response units (RU) for detailed kinetic studies and about 800 RU for concentration studies and screening. Two- or three-fold serial dilutions typically at concentrations spanning 1 μM-0.1 nM (aimed at 0.1-10× estimated K_(D)) of purified Fab fragments were injected for 1 minute at 100 μL/min and dissociation times of 10 minutes were allowed. After each binding cycle, surfaces were regenerated with 25 mM NaOH in 25% v/v ethanol, which was tolerated over hundreds of cycles. Kinetic association rate (k_(on)) and dissociation rate (k_(off)) were obtained simultaneously by fitting the data to a 1:1 Langmuir binding model (Karlsson et al. (1994). Methods Enzymology 6. 99-110) using the BIAevaluation program. Global equilibrium dissociation constants (K_(D)) or “affinities” were calculated from the ratio K_(D)=k_(off)/k_(on). Affinities of the murine Fab fragments are shown in Tables 2 and 3.

Epitope mapping of the murine anti-CGRP antibodies. To determine the epitope that anti-CGRP antibodies bind on human α-CGRP, binding affinities of the Fab fragments to various CGRP fragments were measured as described above by capturing N-terminally biotinylated CGRP fragments amino acids 19-37 and amino acids 25-37 on a SA sensor chip. FIG. 1 shows their binding affinities measured at 25° C. As shown in FIG. 1, all antibodies, except antibody 4901, bind to human α-CGRP fragments 19-37 and 25-37 with affinity similar to their binding affinity to full length human α-CGRP (1-37). Antibody 4901 binds to human α-CGRP fragment 25-37 with six-fold lower affinity than binding to full length human α-CGRP fragment, due mainly to a loss in off-rate. The data indicate that these anti-CGRP antibodies generally bind to the C-terminal end of CGRP.

Alanine scanning was performed to further characterize amino acids in human α-CGRP involved in binding of anti-CGRP antibodies. Different variants of human α-CGRP with single alanine substitutions were generated by peptide synthesis. Their amino acid sequences are shown in Table 3 along with all the other peptides used in the Biacore analysis. Affinities of Fab fragments of the anti-CGRP antibodies to these variants were determined using Biacore as described above. As shown in FIG. 1, all 12 antibodies target a C-terminal epitope, with amino acid F37 being the most crucial residue. Mutation of F37 to alanine significantly lowered the affinity or even completely knocked out binding of the anti-CGRP antibodies to the peptide. The next most important amino acid residue is G33, however, only the high affinity antibodies (7E9, 8B6, 10A8, and 7D11) were affected by alanine replacement at this position. Amino acid residue S34 also plays a significant, but lesser, role in the binding of these four high affinity antibodies.

TABLE 1 Characteristics of the anti-CGRP monoclonal antibodies' binding to human α-CGRP and their antagonist activity Cell-based blocking IC₅₀ (nM human α-CGRP binding sites) KD to K_(D) to binding to at 25° C. (room human human its receptor temp.) α-CGRP α-CGRP at 25° C. measured in at 25° C. at 37° C. (measured by radioligand Antibodies (nM) (nM) cAMP activation) binding assay. 7E9 1.0 0.9 Yes 2.5 8B6 1.1 1.2 Yes 4.0 10A8 2.1 3.0 Yes n.d. 7D11 4.4 5.4 Yes n.d. 6H2 9.3 42 Yes 12.9 4901 61 139 Yes 58 14E10 80 179 Yes n.d. 9B8 85 183 No n.d. 13C2 94 379 No n.d. 14A9 148 581 No n.d. 6D5 210 647 No n.d. 1C5 296 652 No n.d. Note: Antibody 4901 is commercially available (Sigma, Product No. C7113). n.d. = not determined

TABLE 2 Characteristics of the anti-CGRP monoclonal antibodies' binding to rat α-CGRP and antagonist activity Cell-based blocking of binding of rat α-CGRP In vivo K_(D) to rat to its receptor at blocking α-CGRP at 25° C. (measured by in saphenous Antibodies 37° C. (nM) cAMP activation) nerve assay 4901 3.4 Yes Yes 7E9 47 Yes Yes 6H2 54 No No 8B6 75 Yes Yes 7D11 218 Yes Yes 10A8 451 No n.d. 9B8 876 No n.d. 14E10 922 No n.d. 13C2 >1000 No n.d. 14A9 >1000 No n.d. 6D5 >1000 No n.d. 1C5 >1000 No n.d. “n.d.” indicates no test was performed for the antibody.

TABLE 3 Amino acid sequences of human α-CGRP fragments (SEQ ID NOS: 15-40) and related peptides (SEQ ID NOS: 41-47). All peptides are C-terminally amidated except SEQ ID NOS: 36-40. Residues in bold indicate point mutations. CGRP Amino acid sequence SEQ ID NO 1-37 (WT) ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF 15 8-37 VTHRLAGLLSRSGGVVKNNFVPTNVGSKAF 16 19-37 SGGVVKNNFVPTNVGSKAF 17 P29A (19-37) SGGVVKNNFVATNVGSKAF 18 K35A (19-37) SGGVVKNNFVPTNVGSAAF 19 K35E (19-37) SGGVVKNNFVPTNVGSEAF 20 K35M (19-37) SGGVVKNNFVPTNVGSMAF 21 K35Q (19-37) SGGVVKNNFVPTNVGSQAF 22 F37A (19-37) SGGVVKNNFVPTNVGSKAA 23 25-38A NNFVPTNVGSKAFA 24 25-37 NNFVPTNVGSKAF 25 F27A (25-37) NNAVPTNVGSKAF 26 V28A (25-37) NNFAPTNVGSKAF 27 P29A (25-37) NNFVATNVGSKAF 28 T30A (25-37) NNFVPANVGSKAF 29 N31A (25-37) NNFVPTAVGSKAF 30 V32A (25-37) NNFVPTNAGSKAF 31 G33A (25-37) NNFVPTNVASKAF 32 S34A (25-37) NNFVPTNVGAKAF 33 F37A (25-37) NNFVPTNVGSKAA 34 26-37 NFVPTNVGSKAF 35 19-37-COOH SGGVVKNNFVPTNVGSKAF 36 19-36-COOH SGGVVKNNFVPTNVGSKA 37 1-36-COOH ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKA 38 1-19-COOH ACDTATCVTHRLAGLLSRS 39 1-13 -COOH ACDTATCVTHRLA 40 rat α (1-37) SCNTATCVTHRLAGLLSRSGGVVKDNFVPTNVGSEAF 41 rat α (19-37) SGGVVKDNFVPTNVGSEAF 42 human β (1-37) ACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF 43 rat β (1-37) SCNTATCVTHRLAGLLSRSGGVVKDNFVPTNVGSKAF 44 Human calcitonin (1-32) CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP 45 Human amylin (1-37) KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY 46 Human adrenomedullin YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGY 47 (1-52)

Example 2: Screening of Anti-CGRP Antagonist Antibodies Using In Vitro Assays

Murine anti-CGRP antibodies were further screened for antagonist activity in vitro using cell based cAMP activation assay and binding assay.

Antagonist activity measured by cAMP assay. Five microliters of human or rat α-CGRP (final concentration 50 nM) in the presence or absence of an anti-CGRP antibody (final concentration 1-3000 nM), or rat α-CGRP or human α-CGRP (final concentration 0.1 nM-10 μM; as a positive control for c-AMP activation) was dispensed into a 384-well plate (Nunc, Cat. No. 264657). Ten microliters of cells (human SK-N-MC if human α-CGRP is used, or rat L6 from ATCC if rat α-CGRP is used) in stimulation buffer (20 mM HEPES, pH 7.4, 146 mM NaCl, 5 mM KCl, 1 mM CaCl₂), 1 mM MgCl₂, and 500 μM 3-Isobutyl-1-methylxanthine (IBMX)) were added into the wells of the plate. The plate was incubated at room temperature for 30 minutes.

After the incubation, cAMP activation was performed using HitHunter™ Enzyme Fragment Complementation Assay (Applied Biosystems) following manufacture's instruction. The assay is based on a genetically engineered β-galactosidase enzyme that consists of two fragments—termed Enzyme Acceptor (EA) and Enzyme Donor (ED). When the two fragments are separated, the enzyme is inactive. When the fragments are together they can recombine spontaneously to form active enzyme by a process called complementation. The EFC assay platform utilizes an ED-cAMP peptide conjugate in which cAMP is recognized by anti-cAMP. This ED fragment is capable of reassociation with EA to form active enzyme. In the assay, anti-cAMP antibody is optimally titrated to bind ED-cAMP conjugate and inhibit enzyme formation. Levels of cAMP in cell lysate samples compete with ED-cAMP conjugate for binding to the anti-cAMP antibody. The amount of free ED conjugate in the assay is proportional to the concentration of cAMP. Therefore, cAMP is measured by the formation of active enzyme that is quantified by the turnover of β-galactosidase luminescent substrate. The cAMP activation assay was performed by adding 10 μl of lysis buffer and anti-cAMP antibody (1:1 ratio) following by incubation at room temperature for 60 min. Then 10 μl of ED-cAMP reagent was added into each well and incubated for 60 minutes at room temperature. After the incubation, 20 μl of EA reagent and CL mixture (containing the substrate) (1:1 ratio) was added into each well and incubated for 1-3 hours or overnight at room temperature. The plate was read at 1 second/well on PMT instrument or 30 seconds/place on imager. The antibodies that inhibit activation of cAMP by α-CGRP were identified (referred to as “yes”) in Tables 2 and 3 above. Data in Tables 2 and 3 indicate that antibodies that demonstrated antagonist activity in the assay generally have high affinity. For example, antibodies having K_(D) (determined at 25° C.) of about 80 nM or less to human α-CGRP or having K_(D) (determined at 37° C.) of about 47 nM or less to rat α-CGRP showed antagonist activity in this assay.

Radioligand binding assay. Binding assay was performed to measure the IC₅₀ of anti-CGRP antibody in blocking the CGRP from binding to the receptor as described previously. Zimmermann et al., Peptides 16:421-4, 1995; Mallee et al., J. Biol. Chem. 277:14294-8, 2002. Membranes (25 Kg) from SK-N-MC cells were incubated for 90 min at room temperature in incubation buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl₂, 0.1% BSA) containing 10 pM ¹²⁵I-human α-CGRP in a total volume of 1 mL. To determine inhibition concentrations (IC₅₀), antibodies or unlabeled CGRP (as a control), from a about 100 fold higher stock solution were dissolved at varying concentrations in the incubation buffer and incubated at the same time with membranes and 10 pM ¹²⁵I-human α-CGRP. Incubation was terminated by filtration through a glass microfiber filter (GF/B, 1 μm) which had been blocked with 0.5% polyethylemimine. Dose response curves were plotted and K_(i) values were determined by using the equation: K_(i)=IC₅₀/(1+([ligand]/K_(D)); where the equilibrium dissociation constant K_(D)=8 pM for human α-CGRP to CGRP1 receptor as present in SK-N-MC cells, and B_(max)=0.025 pmol/mg protein. The reported IC₅₀ value (in terms of IgG molecules) was converted to binding sites (by multiplying it by 2) so that it could be compared with the affinities (K_(D)) determined by Biacore (see Table 1).

Table 1 shows the IC₅₀ of murine antibodies 7E9, 8B6, 6H2 and 4901. Data indicate that antibody affinity generally correlates with IC₅₀: antibodies with higher affinity (lower K_(D) values) have lower IC₅₀ in the radioligand binding assay.

Example 3: Effect of Anti-CGRP Antagonist Antibodies on Skin Vasodilatation Induced by Stimulation of Rat Saphenous Nerve

To test antagonist activity of anti-CGRP antibodies, effect of the antibodies on skin vasodilatation by stimulation of rat saphenous nerve was tested using a rat model described previously. Escott et al., Br. J. Pharmacol. 110:772-776, 1993. In this rat model, electrical stimulation of saphenous nerve induces release of CGRP from nerve endings, resulting in an increase in skin blood flow. Blood flow in the foot skin of male Sprague Dawley rats (170-300 g, from Charles River Hollister) was measured after saphenous nerve stimulation. Rats were maintained under anesthesia with 2% isoflurane. Bretylium tosylate (30 mg/kg, administered i.v.) was given at the beginning of the experiment to minimize vasoconstriction due to the concomitant stimulation of sympathetic fibers of the saphenous nerve. Body temperature was maintained at 37° C. by the use of a rectal probe thermostatically connected to a temperature controlled heating pad. Compounds including antibodies, positive control (CGRP 8-37), and vehicle (PBS, 0.01% Tween 20) were given intravenously through the right femoral vein, except for the experiment shown in FIG. 3, the test compound and the control were injected through tail vein, and for experiments shown in FIGS. 2A and 2B, antibodies 4901 and 7D11 were injected intraperitoneally (IP). Positive control compound CGRP 8-37 (vasodilatation antagonist), due to its short half-life, was given 3-5 min before nerve stimulation at 400 nmol/kg (200 μl). Tan et al., Clin. Sci. 89:656-73, 1995. The antibodies were given in different doses (1 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, and 25 mg/kg).

For experiments shown in FIGS. 2A and 2B, antibody 4901 (25 mg/kg), antibody 7D11 (25 mg/kg), or vehicle control (PBS with 0.01% Tween 20) was administered intraperitoneally (IP) 72 hours before the electrical pulse stimulation. For experiment shown in FIG. 3, antibody 4901 (1 mg/kg, 2.5 mg/kg, 5 mg/kg, or 25 mg/kg) or vehicle control (PBS with 0.01% Tween 20) was administered intravenously 24 hours before the electrical pulse stimulation. After administration of the antibodies or vehicle control, the saphenous nerve of the right hindlimb was exposed surgically, cut proximally and covered with plastic wrap to prevent drying. A laser Doppler probe was placed over the medio-dorsal side of the hindpaw skin, which is the region innervated by the saphenous nerve. Skin blood flow, measured as blood cell flux, was monitored with a laser Doppler flow meter. When a stable base-line flux (less than 5% variation) was established for at least 5 minutes, the nerve was placed over platinum bipolar electrodes and electrically stimulated with 60 pulses (2 Hz, 10 V, 1 ms, for 30 seconds) and then again 20 minutes later. Cumulative change in skin blood flow was estimated by the area under the flux-time curve (AUC, which is equal to change in flux multiplied by change in time) for each flux response to electrical pulse stimulation. The average of the blood flow response to the two stimulations was taken. Animals were kept under anesthesia for a period of one to three hours.

As shown in FIG. 2A and FIG. 2B, blood flow increase stimulated by applying electronic pulses on saphenous nerve was inhibited by the presence of CGRP 8-37 (400 nmol/kg, administered i.v.), antibody 4901 (25 mg/kg, administered ip), or antibody 7D11 (25 mg/kg, administered ip) as compared to the control. CGRP 8-37 was administered 3-5 minutes before the saphenous nerve stimulation; and antibodies were administered 72 hours before the saphenous nerve stimulation. As shown in FIG. 3, blood flow increase stimulated by applying electronic pulses on saphenous nerve was inhibited by the presence of antibody 4901 at different doses (1 mg/kg, 2.5 mg/kg, 5 mg/kg, and 25 mg/kg) administered intravenously at 24 hours before the saphenous nerve stimulation.

For experiments shown in FIGS. 4A and 4B, saphenous nerve was exposed surgically before antibody administration. The saphenous nerve of the right hindlimb was exposed surgically, cut proximally and covered with plastic wrap to prevent drying. A laser Doppler probe was placed over the medio-dorsal side of the hindpaw skin, which is the region innervated by the saphenous nerve. Skin blood flow, measured as blood cell flux, was monitored with a laser Doppler flow meter. Thirty to forty-five minutes after bretylium tosylate injection, when a stable base-line flux (less than 5% variation) was established for at least 5 minutes, the nerve was placed over platinum bipolar electrodes and electrically stimulated (2 Hz, 10V, 1 ms, for 30 seconds) and again 20 minutes later. The average of the blood flow flux response to these two stimulations was used to establish the baseline response (time 0) to electrical stimulation. Antibody 4901 (1 mg/kg or 10 mg/kg), antibody 7E9 (10 mg/kg), antibody 8B6 (10 mg/kg), or vehicle (PBS with 0.01% Tween 20) were then administered intravenously (i.v.). The nerve was subsequently stimulated (2 Hz, 10V, 1 ms, for 30 sec) at 30 minutes, 60 minutes, 90 minutes, and 120 minutes after antibody or vehicle administration. Animals were kept under anesthesia for a period of approximately three hours. Cumulative change in skin blood flow was estimated by the area under the flux-time curve (AUC, which is equal to change in flux multiplied by change in time) for each flux response to electrical pulse stimulations.

As shown in FIG. 4A, blood flow increase stimulated by applying electronic pulses on saphenous nerve was significantly inhibited by the presence of antibody 4901 1 mg/kg administered i.v., when electronic pulse stimulation was applied at 60 minutes, 90 minutes, and 120 minutes after the antibody administration, and blood flow increase stimulated by applying electronic pulses on saphenous nerve was significantly inhibited by the presence of antibody 4901 10 mg/kg administered i.v., when electronic pulse stimulation was applied at 30 minutes, 60 minutes, 90 minutes, and 120 minutes after the antibody administration. FIG. 4B shows that blood flow increase stimulated by applying electronic pulses on saphenous nerve was significantly inhibited by the presence of antibody 7E9 (10 mg/kg, administered i.v.) when electronic pulse stimulation was applied at 30 min, 60 min, 90 min, and 120 min after antibody administration, and by the presence of antibody 8B6 (10 mg/kg, administered i.v.) when electronic pulse stimulation was applied at 30 min after antibody administration.

These data indicate that antibodies 4901, 7E9, 7D11, and 8B6 are effective in blocking CGRP activity as measured by skin vasodilatation induced by stimulation of rat saphenous nerve.

Example 4. Characterization of Anti-CGRP Antibody G1 and its Variants

Amino acid sequences for the heavy chain variable region and light chain variable region of anti-CGRP antibody G1 are shown in FIG. 5. The following methods were used for expression and characterization of antibody G1 and its variants.

Expression vector used. Expression of the Fab fragment of the antibodies was under control of an IPTG inducible lacZ promoter similar to that described in Barbas (2001) Phage display: a laboratory manual, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press pg. 2.10. Vector pComb3X), however, modifications included addition and expression of the following additional domains: the human Kappa light chain constant domain and the CH1 constant domain of IgG2 human immunoglobulin, Ig gamma-2 chain C region, protein accession number P01859; Immunoglobulin kappa light chain (Homo sapiens), protein accession number CAA09181.

Small scale Fab preparation. From E. coli transformed (either using electroporation-competent TG1 cells or chemically-competent Top 10 cells) with a Fab library, single colonies were used to inoculate both a master plate (agar LB+carbenicillin (50 μg/mL)+2% glucose) and a working plate (2 mL/well, 96-well/plate) where each well contained 1.5 mL LB+carbenicillin (50 μg/mL)+2% glucose. A gas permeable adhesive seal (ABgene, Surrey, UK) was applied to the plate. Both plates were incubated at 30° C. for 12-16 hours; the working plate was shaken vigorously. The master plate was stored at 4° C. until needed, while the cells from the working plate were pelleted (4000 rpm, 4° C., 20 minutes) and resuspended in 1.0 mL LB+carbenicillin (50 μg/mL)+0.5 mM IPTG to induce expression of Fabs by vigorous shaking for 5 hours at 30° C. Induced cells were centrifuges at 4000 rpm, 4° C. for 20 minutes and resuspended in 0.6 mL Biacore HB-SEP buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% v/v P20). Lysis of HB-SEP resuspended cells was accomplished by freezing (−80° C.) and then thawing at 37° C. Cell lysates were centrifuged at 4000 rpm, 4° C. for 1 hour to separate the debris from the Fab-containing supernatants, which were subsequently filtered (0.2 μm) using a Millipore MultiScreen Assay System 96-Well Filtration Plate and vacuum manifold. Biacore was used to analyze filtered supernatants by injecting them across CGRPs on the sensor chip. Affinity-selected clones expressing Fabs were rescued from the master plate, which provided template DNA for PCR, sequencing, and plasmid preparation.

Large scale Fab preparation. To obtain kinetic parameters, Fabs were expressed on a larger scale as follows. Erlenmeyer flasks containing 150 mL LB+carbenicillin (50 μg/mL)+2% glucose were inoculated with 1 mL of a “starter” overnight culture from an affinity-selected Fab-expressing E. coli clone. The remainder of the starter culture (˜3 mL) was used to prepare plasmid DNA (QIAprep mini-prep, Qiagen kit) for sequencing and further manipulation. The large culture was incubated at 30° C. with vigorous shaking until an OD_(600 nm) of 1.0 was attained (typically 12-16 h). The cells were pelleted by centrifuging at 4000 rpm, 4° C. for 20 minutes, and resuspended in 150 mL LB+carbenicillin (50 μg/mL)+0.5 mM IPTG. After 5 hours expression at 30° C., cells were pelleted by centrifuging at 4000 rpm, 4° C. for 20 minutes, resuspended in 10 mL Biacore HBS-EP buffer, and lysed using a single freeze (−80° C.)/thaw (37° C.) cycle. Cell lysates were pelleted by centrifuging at 4000 rpm, 4° C. for one hour, and the supernatant was collected and filtered (0.2 um). Filtered supernatants were loaded onto Ni-NTA superflow sepharose (Qiagen, Valencia, Calif.) columns equilibrated with PBS, pH 8, then washed with 5 column volumes of PBS, pH 8. Individual Fabs eluted in different fractions with PBS (pH 8)+300 mM Imidazole. Fractions containing Fabs were pooled and dialyzed in PBS, then quantified by ELISA prior to affinity characterization.

Full antibody preparation. For expression of full antibodies, heavy and light chain variable regions were cloned in mammalian expression vectors and transfected using lipofectamine into HEK 293 cells for transient expression. Antibodies were purified using protein A using standard methods.

Vector pDb.CGRP.hFcGI is an expression vector comprising the heavy chain of the G1 antibody, and is suitable for transient or stable expression of the heavy chain. Vector pDb.CGRP.hFcGI has nucleotide sequences corresponding to the following regions: the murine cytomegalovirus promoter region (nucleotides 7-612); a synthetic intron (nucleotides 613-1679); the DHFR coding region (nucleotides 688-1253); human growth hormone signal peptide (nucleotides 1899-1976); heavy chain variable region of G1 (nucleotides 1977-2621); human heavy chain IgG2 constant region containing the following mutations: A330P331 to S330S331 (amino acid numbering with reference to the wildtype IgG2 sequence; see Eur. J. Immunol. (1999) 29:2613-2624). Vector pDb.CGRP.hFcGI was deposited at the ATCC on Jul. 15, 2005, and was assigned ATCC Accession No. PTA-6867.

Vector pEb.CGRP.hKGI is an expression vector comprising the light chain of the G1 antibody, and is suitable for transient expression of the light chain. Vector pEb.CGRP.hKGI has nucleotide sequences corresponding to the following regions: the murine cytomegalovirus promoter region (nucleotides 2-613); human EF-1 intron (nucleotides 614-1149); human growth hormone signal peptide (nucleotides 1160-1237); antibody G1 light chain variable region (nucleotides 1238-1558); human kappa chain constant region (nucleotides 1559-1882). Vector pEb.CGRP.hKGI was deposited at the ATCC on Jul. 15, 2005, and was assigned ATCC Accession No. PTA-6866.

Biacore assay for affinity determination. Affinities of G1 monoclonal antibody and its variants were determined at either 25° C. or 37° C. using the BIACORE3000™ surface plasmon resonance (SPR) system (Biacore, INC, Piscataway N.J.). Affinity was determined by capturing N-terminally biotinylated CGRP or fragments via pre-immobilized streptavidin (SA sensor chip) and measuring the binding kinetics of antibody G1 Fab fragments or variants titrated across the CGRP or fragment on the chip. All Biacore assays were conducted in HBS-EP running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% v/v polysorbate P20). CGRP surfaces were prepared by diluting the N-biotinylated CGRP to a concentration of less than 0.001 mg/mL into HBS-EP buffer and injecting it across the SA sensor chip using variable contact times. Low capacity surfaces, corresponding to capture levels <50 response units (RU) were used for high-resolution kinetic studies, whereas high capacity surfaces (about 800 RU of captured CGRP) were used for concentration studies, screening, and solution affinity determinations. Kinetic data were obtained by diluting antibody G1 Fab serially in two- or three-fold increments to concentrations spanning 1 uM-0.1 nM (aimed at 0.1-10× estimated K_(D)). Samples were typically injected for 1 minute at 100 μL/min and dissociation times of at least 10 minutes were allowed. After each binding cycle, surfaces were regenerated with 25 mM NaOH in 25% v/v ethanol, which was tolerated over hundreds of cycles. An entire titration series (typically generated in duplicate) was fit globally to a 1:1 Langmuir binding model using the BIAevaluation program. This returned a unique pair of association and dissociation kinetic rate constants (respectively, k_(on) and k_(off)) for each binding interaction, whose ratio gave the equilibrium dissociation constant (K_(D)=k_(off)/k_(on)). Affinities (K_(D) values) determined in this way are listed in Tables 6 and 7.

High-resolution analysis of binding interactions with extremely slow off rates. For interactions with extremely slow off rates (in particular, antibody G1 Fab binding to human α-CGRP on the chip at 25° C.), affinities were obtained in a two-part experiment. The protocol described above was used with the following modifications. The association rate constant (k_(on)) was determined by injecting a 2-fold titration series (in duplicate) spanning 550 nM-1 nM for 30 seconds at 100 μL/min and allowing only a 30 second dissociation phase. The dissociation rate constant (k_(off)) was determined by injecting three concentrations (high, medium, and low) of the same titration series in duplicate for 30 seconds and allowing a 2-hour dissociation phase. The affinity (K_(D)) of each interaction was obtained by combining the k_(on) and k_(off) values obtained in both types of experiments, as shown in Table 4.

Determining solution affinity by Biacore. The solution affinity of antibody G1 for rat α-CGRP and F37A (19-37) human α-CGRP was measured by Biacore at 37° C. A high capacity CGRP chip surface was used (the high-affinity human α-CGRP was chosen for detection purposes) and HBS-EP running buffer was flowed at 5 μL/min. Antibody G1 Fab fragment at a constant concentration of 5 nM (aimed to be at or below the expected K_(D) of the solution-based interaction) was pre-incubated with competing peptide, either rat α-CGRP or F37A (19-37) human α-CGRP, at final concentrations spanning 1 nM to 1 μM in 3-fold serial dilutions. Antibody G1 Fab solutions in the absence or presence of solution-based competing peptide, were injected across CGRP on the chip and the depletion of binding responses detected at the chip surface as a result of solution competition was monitored. These binding responses were converted to “free Fab concentrations” using a calibration curve, which was constructed by titrating antibody G1 Fab alone (5, 2.5, 1.25, 0.625, 0.325 and 0 nM) across the CGRP on the chip. “Free Fab concentrations” were plotted against the concentration of competing solution-based peptide used to generate each data point and fit to a solution affinity model using the BIAevaluation software. The solution affinities determined (indirectly) in this way are shown in Tables 4 and 6 and were used to validate the affinities obtained when Fabs are injected directly across N-biotinylated CGRPs on a SA chip. The close agreement between the affinities determined by these two methods confirms that tethering an N-biotinylated version of the CGRP to the chip does not alter its native solution binding activity.

Table 4 below shows the binding affinities of antibody G1 to human α-CGRP, human β-CGRP, rat α-CGRP, and rat β-CGRP determined by Biacore, by flowing Fab fragments across N-biotinylated CGRPs on a SA chip. To better resolve the affinities of binding interactions with extremely slow offrates, affinities were also determined in a two-part experiment to complement this assay orientation, the solution affinity of the rat α-CGRP interaction was also determined (as described above). The close agreement of the affinities measured in both assay orientations confirms that the binding affinity of the native rat α-CGRP in solution is not altered when it is N-biotinylated and tethered to a SA chip.

TABLE 4 Binding affinities of antibody G1 Fabs titrated across CGRPs on the chip Temp. CGRP on chip (° C.) k_(on) (1/Ms) k_(off) (1/s) K_(D) (nM) Human α-CGRP 25 1.86 × 10⁵ 7.80 × 10⁻⁶ 0.042 (7%, n = 4)* Human α-CGRP 37 5.78 × 10⁵ 3.63 × 10⁻⁵ 0.063 (4%, n = 2)* Human β-CGRP 37 4.51 × 10⁵ 6.98 × 10⁻⁵ 0.155 Rat α-CGRP 25 5.08 × 10⁴ 6.18 × 10⁻⁵  1.22 (12%, n = 2)* Rat α-CGRP 37 1.55 × 10⁵ 3.99 × 10⁻⁴ 2.57* (Solution K_(D) = 10 (50%, n = 4)** Rat β-CGRP 37 5.16 × 10⁵ 7.85 × 10⁻⁵ 0.152  *Affinities for α-CGRPs (rat and human) were determined in a high-resolution two-part experiment, in which the dissociation phase was monitored for 2 hours (the values for k_(on), k_(off), and K_(D) represent the average of n replicate experiments with the standard deviation expressed as a percent variance). Affinities for β-CGRPs (rat and human) were determined by global analysis using only a 20-min dissociation phase, which was not accurate enough to quantify their extremely off-rates (their off-rates are likely slower than stated here and therefore their affinities are likely even higher). Antibody G1 Fab dissociated extremely slowly from all CGRPs (except α-rat CGRP) with off-rates that approached the resolution limit of the Biacore assay (especially at 25° C.). **Solution affinity determined by measuring the depletion of binding responses detected at CGRP on the chip for antibody G1 Fab pre-incubated with solution-based rat α-CGRP competitor.

Table 5 below shows antibodies having the amino acid sequence variation as compared to antibody G1 and their affinities to both rat α-CGRP and human α-CGRP. All amino acid substitutions of the variants shown in Table 5 are described relative to the sequence of G1. The binding affinities of Fab fragments were determined by Biacore by flowing them across CGRPs on a SA chip.

TABLE 5 Amino acid sequences and binding affinity data for antibody G1 variants determined at 37° C. by Biacore. α-rat α-rat α-human α-human Clone L1 L2 H2 HC-FW3 k_(off) (1/s) K_(D) (nM) k_(off) (1/s) K_(D) (nM) G1 3.99 × 10⁻⁴   2.57 3.63 × 10⁻⁵  0.063 M1 A100L 1.10 × 10⁻³ 1.73 × 10⁻⁴ M2 L99A  2.6 × 10⁻³ 58   3.1 × 10⁻⁴ 3   A100R M3 L99A  2.0 × 10⁻³ 61   2.1 × 10⁻⁴ 1.7  A100S M4 L99A 1.52 × 10⁻³  84.4 6.95 × 10⁻⁵ 0.43 A100V M5 L99A 7.35 × 10⁻⁴  40.8 3.22 × 10⁻⁵ 0.20 A100Y M6 L99N 7.84 × 10⁻⁴  43.6 1.33 × 10⁻⁴ 0.83 M7 L99N 9.18 × 10⁻⁴  51.0 2.43 × 10⁻⁴ 1.52 A100C M8 L99N 7.45 × 10⁻⁴  41.4 9.20 × 10⁻⁵ 0.58 A100G M9 L99N n.d. n.d. 1.00 × 10⁻⁵ 0.06 A100Y M10 L99S 1.51 × 10⁻³  83.9 1.73 × 10⁻⁴ 1.08 A100S M11 L99S 4.83 × 10⁻³ 268.3 2.83 × 10⁻⁴ 1.77 A100T M12 L99S 1.94 × 10⁻³ 107.8 1.01 × 10⁻⁴ 0.63 A100V M13 L99T 1.84 × 10⁻³ 102.2 1.86 × 10⁻⁴ 1.16 A100G M14 L99T n.d. n.d. 1.00 × 10⁻⁵ 0.06 A100K M15 L99T 1.15 × 10⁻³  63.9 1.58 × 10⁻⁵ 0.10 A100P M16 L99T 9.96 × 10⁻⁴  55.3 1.65 × 10⁻⁴ 1.03 A100S M17 L99T 2.06 × 10⁻³ 114.4 1.85 × 10⁻⁴ 1.16 A100V M18 L99V 1.22 × 10⁻³  67.8 7.03 × 10⁻⁵ 0.44 A100G M19 L99V n.d. n.d. 1.00 × 10⁻⁵ 0.06 A100R M20 R28W L99R 1.44 × 10⁻³  80.0 1.36 × 10⁻⁴ 0.85 A100L M21 R28W L99S 6.95 × 10⁻⁴  15.2 1.42 × 10⁻⁴ 1.23 M22 R28W L99T 1.10 × 10⁻³  61.1 1.16 × 10⁻⁴ 0.73 M23 R28G L99T 7.99 × 10⁻⁴  44.4 1.30 × 10⁻⁴ 0.81 A100V M24 R28L L99T 1.04 × 10⁻³  57.8 1.48 × 10⁻⁴ 0.93 A100V M25 R28N L99T  1.4 × 10⁻³ 76   1.4 × 10⁻ ⁴ 1.3  A100V M26 R28N A57G L99T 9.24 × 10⁻⁴  51.3 1.48 × 10⁻⁴ 0.93 A100V M27 R28N L99T 3.41 × 10⁻³ 189.4 3.57 × 10⁻⁴ 2.23 T30A A100V M28 R28N E54R L99T 1.25 × 10⁻³  69.4 9.96 × 10⁻⁵ 0.62 T30D A57N A100V M29 R28N L99T 3.59 × 10⁻³ 199.4 3.80 × 10⁻⁴ 2.38 T30G A100V M30 R28N E54K L99T 6.38 × 10⁻³ 354.4 5.90 × 10⁻⁴ 3.69 T30G A57E A100V M31 R28N E54K L99T 3.61 × 10⁻³ 200.6 3.47 × 10⁻⁴ 2.17 T30G A57G A100V M32 R28N E54K L99T 2.96 × 10⁻³ 164.4 2.71 × 10⁻⁴ 1.69 T30G A57H A100V M33 R28N E54K L99T 9.22 × 10⁻³ 512.2 7.50 × 10⁻⁴ 4.69 T30G A57N A100V S58G M34 R28N E54K L99T 2.17 × 10⁻³ 120.6 6.46 × 10⁻⁴ 4.04 T30G A57N A100V S58T M35 R28N E54K L99T 3.99 × 10⁻³ 221.7 3.39 × 10⁻⁴ 2.12 T30G A57S A100V M36 R28N L99T 4.79 × 10⁻³ 266.1 2.39 × 10⁻⁴ 1.49 T30R A100V M37 R28N A57G L99T 1.45 × 10⁻³  80.6 2.26 × 10⁻⁴ 1.41 T30S A100V M38 R28N L99T 5.11 × 10⁻³ 283.9 2.18 × 10⁻⁴ 1.36 T30W A100V M39 R28N G50A A57N L99T 9.95 × 10⁻³ 552.8 4.25 × 10⁻⁴ 2.66 L56T S58Y A100V M40 R28N G50A E54K L99T 0.36  20000.0  1.28 × 10⁻³ 8.00 L56T A57L A100V M41 R28N G50A E54K L99T 4.53 × 10⁻³ 251.7 2.10 × 10⁻⁴ 1.31 L56T A57N A100V E64D M42 R28N G50A E54K L99T 7.52 × 10⁻³ 417.8 4.17 × 10⁻⁴ 2.61 L56T A57N A100V H61F M43 R28N G50A E54K L99T 4.53 × 10⁻³ 251.7 2.63 × 10⁻⁴ 1.64 L56T A57N A100V S58C M44 R28N G50A E54K L99T 6.13 × 10⁻³ 443   2.10 × 10⁻⁴ 2.05 L56T A57N A100V S58E M45 R28N G50A E54K L99T 5.58 × 10⁻³ 259   2.11 × 10⁻⁴ 1.85 L56T A57N A100V S58E E64D M46 R28N G50A E54K L99T 2.94 × 10⁻³ 163.3 5.39 × 10⁻⁴ 3.37 L56T A57N A100V S58E H61F M47 R28N G50A E54K L99T 8.23 × 10⁻³ 457.2 3.32 × 10⁻⁴ 2.08 L56T A57N A100V S58G M48 R28N G50A E54K L99T 0.0343 1905.6  8.42 × 10⁻⁴ 5.26 L56T A57N A100V S58L M49 R28N G50A E54K L99T 0.0148 822.2 5.95 × 10⁻⁴ 3.72 L56T A57N A100V S58Y H61F M50 R28N G50A E54K L99T 5.30 × 10⁻³ 294.4 4.06 × 10⁻⁴ 2.54 L56T A57R A100V M51 R28N L561 E54K L99T 1.18 × 10⁻³  65.6 1.31 × 10⁻⁴ 0.82 A57G A100V M52 R28N L561 E54K L99T 2.29 × 10⁻³ 127.2 2.81 × 10⁻⁴ 1.76 A57N A100V S58A M53 R28N L561 E54K L99T 1.91 × 10⁻³ 106.1 3.74 × 10⁻⁴ 2.34 A57N A100V S58G M54 R28N G50A E54K L99T 2.16 × 10⁻³ 120.0 1.79 × 10⁻³ 11.19  T30A A57N A100V S58P M55 R28N L56S E54K L99T 5.85 × 10⁻³ 325.0 4.78 × 10⁻⁴ 2.99 T30A A57N A100V S58E E64D M56 R28N L56S E54K L99T 9.35 × 10⁻³ 519.4 4.79 × 10⁻⁴ 2.99 T30D A57N A100V H61F M57 R28N L56S E54K L99T 0.0104 1,200   3.22 × 10⁻⁴ 3.08 T30D A57N A100V S58E M58 R28N L56S E54K L99T No binding n.d. 1.95 × 10⁻³ 12.19  T30D A57N A100V S58I H61F M59 R28N L56S E54K L99T 0.0123 683.3 5.24 × 10⁻⁴ 3.28 T30D A57N A100V S58N H61F M60 R28N L56S E54K L99T 0.0272 1511.1  9.11 × 10⁻⁴ 5.69 T30D A57N A100V S58R H61F M61 R28N A51H E54Q L99T 5.21 × 10⁻³ 289.4 4.59 × 10⁻⁴ 2.87 T30G A57N A100V H61F M62 R28N A51H E54K L99T 5.75 × 10⁻³ 242   5.57 × 10⁻⁴ 5.86 T30G L56T A57N A100V S58E M63 R28N G50A E54K L99T 2.65 × 10⁻³ 147.2 1.50 × 10⁻³ 9.38 T30G A57N A100V S58T M64 R28N G50A E54K L99T 0.0234 1300.0  1.32 × 10⁻³ 8.25 T30G A57N A100V S58V M65 R28N G50A E54K L99T 4.07 × 10⁻³ 226.1 8.03 × 10⁻⁴ 5.02 T30G L56I A57C A100V M66 R28N L561 E54K L99T 5.11 × 10⁻³ 283.9 5.20 × 10⁻⁴ 3.25 T30G A57E A100V M67 R28N L561 E54K L99T 1.71 × 10⁻³  95.0 8.20 × 10⁻⁴ 5.13 T30G A57F A100V M68 R28N L561 E54K L99T 6.76 × 10⁻³ 375.6 4.28 × 10⁻⁴ 2.68 T30G A57N A100V S58D E64D M69 R28N L561 E54K L99T 1.81 × 10⁻³ 100.6 7.33 × 10⁻⁴ 4.58 T30G A57N A100V S58E M70 R28N L561 E54K L99T 6.07 × 10⁻³ 337.2 5.59 × 10⁻⁴ 3.49 T30G A57S A100V M71 R28N L561 E54K L99T 2.12 × 10⁻³ 117.8 1.28 × 10⁻³ 8.00 T30G A57Y A100V M72 R28N L56S E54K L99T 3.95 × 10⁻³ 219.4 4.00 × 10⁻⁴ 2.50 T30G A100V M73 R28N L56S E54K L99T 3.00 × 10⁻³ 166.7 2.55 × 10⁻⁴ 1.59 T30G A57N A100V S58Y E64D M74 R28N L56S E54K L99T 6.03 × 10⁻³ 335.0 5.97 × 10⁻⁴ 3.73 T30G A57S A100V M75 R28N L56S E54K L99T 1.87 × 10⁻² 1038.9  1.16 × 10⁻³ 7.25 T30G A57V A100V M76 R28N G50A A57G L99T 1.16 × 10⁻³  64.4 3.64 × 10⁻⁴ 2.28 T30S L56T A100V M77 R28N G50A E54K L99T 0.0143 794.4 4.77 × 10⁻⁴ 2.98 T30S L56T A57D A100V M78 R28N G50A E54K L99T 0.167  9277.8  1.31 × 10⁻³ 8.19 T30S L56T A57N A100V S58T M79 R28N G50A E54K L99T 0.19  10555.6  1.29 × 10⁻³ 8.06 T30S L56T A57P A100V M80 R28N L561 E54K L99T 0.0993 5516.7  2.09 × 10⁻³ 13.06  T30S A57N A100V S58V M81 R28N L56S E54K L99T 4.29 × 10⁻³ 238.3 4.90 × 10⁻⁴ 3.06 T30S A57N A100V S58E M82 R28N A51H A57N L99T 6.99 × 10⁻³ 388.3 8.77 × 10⁻⁴ 5.48 T30V L56T A100V M83 R28N A51H E54K L99T No binding n.d. 9.33 × 10⁻⁴ 5.83 T30V L56T A57N A100V S58M H61F M84 R28N A51H E54N L99T 1.76 × 10⁻² 977.8 1.08 × 10⁻³ 6.75 T30V L56T A57N A100V

All CDRs including both Kabat and Chothia CDRs. Amino acid residues are numbered sequentially (see FIG. 5). All clones have L3+H1+H3 sequences identical to G1. K_(D)=k_(off)/k_(on). All k_(off) values were determined in a screening mode except those that are underlined, which were obtained by global analysis of a Fab concentration series (G1 was analyzed in a high-resolution mode). Underlined K_(D) values were therefore determined experimentally by measuring k_(on). Other k_(on) values were estimated to be the same as M25. n.d.=not determined

To determine the epitope on human α-CGRP that is recognized by antibody G1, Biacore assays described above were used. Human α-CGRP was purchased as an N-biotinylated version to enable its high-affinity capture via SA sensor chips. The binding of G1 Fab fragment to the human α-CGRP on the chip in the absence or presence of a CGRP peptide was determined. Typically, a 2000:1 mol peptide/Fab solution (e.g., 10 μM peptide in 50 nM G1 Fab) was injected across human α-CGRP on the chip. FIG. 6 shows the percentage of binding blocked by competing peptide. Data shown in FIG. 6 indicate that peptides that block 100% binding of G1 Fab to human α-CGRP are 1-37 (WT), 8-37, 26-37, P29A (19-37), K35A (19-37), K35E (19-37), and K35M (19-37) of human α-CGRP; 1-37 of β-CGRP (WT); 1-37 of rat α-CGRP (WT); and 1-37 of rat β-CGRP (WT). All these peptides are amidated at the C-terminus. Peptides F37A (19-37) and 19-37 (the latter not amidated at the C-terminus) of human α-CGRP also blocked about 80% to 90% of binding of G1 Fab to human α-CGRP. Peptide 1-36 (not amidated at the C-terminus) of human α-CGRP blocked about 40% of binding of G1 Fab to human α-CGRP. Peptide fragment 19-36 (amidated at the C-terminus) of human α-CGRP; peptide fragments 1-13 and 1-19 of human α-CGRP (neither of which are amidated at the C-terminus); and human amylin, calcitonin, and adrenomedullin (all amidated at the C-terminus) did not compete with binding of G1 Fab to human α-CGRP on the chip. These data demonstrate that G1 targets a C-terminal epitope of CGRP and that both the identity of the most terminal residue (F37) and its amidation is important for binding.

Binding affinities of G1 Fab to variants of human α-CGRP (at 37° C.) was also determined. Table 6 below shows the affinities as measured directly by titrating G1 Fab across N-biotinylated human α-CGRP and variants on the chip. Data in Table 6 indicate that antibody G1 binds to a C-terminal epitope with F37 and G33 being the most important residues. G1 does not bind to CGRP when an extra amino acid residue (alanine) is added at the C-terminal (which is amidated).

TABLE 6 Binding affinities of G1 Fab to human α-CGRP and variants measured at 37° C. (see Table 3 for their amino acid sequences) CGRP on chip k_(on) (1/Ms) k_(off) (1/s) K_(D) (nM) 1-37 (WT) 4.68 × 10⁵ 7.63 × 10⁻⁵ 0.16 (high resolution K_(D) = 0.06) 19-37 4.60 × 10⁵ 7.30 × 10⁻⁵ 0.16 25-37 3.10 × 10⁵ 8.80 × 10⁻⁵ 0.28 F27A (25-37) 3.25 × 10⁵ 1.24 × 10⁻⁴ 0.38 V28A (25-37) 3.32 × 10⁵ 9.38 × 10⁻⁵ 0.28 P29A (25-37) 2.26 × 10⁵ 1.78 × 10⁻⁴ 0.79 T30A (25-37) 1.79 × 10⁵ 8.41 × 10⁻⁵ 0.47 N31A (25-37) 2.17 × 10⁵ 1.14 × 10⁻⁴ 0.53 V32A (25-37) 2.02 × 10⁵ 3.46 × 10⁻⁴ 1.71 G33A (25-37) 2.07 × 10⁵ 0.0291 141 S34A (25-37) 2.51 × 10⁵ 7.64 × 10⁻⁴ 3.04 K35A (19-37) 2.23 × 10⁵ 2.97 × 10⁻⁴ 1.33 K35E (19-37) 5.95 × 10⁴ 5.79 × 10⁻⁴ 9.73 K35M (19-37) 2.63 × 10⁵ 1.34 × 10⁻⁴ 0.51 K35Q (19-37) 1.95 × 10⁵ 2.70 × 10⁻⁴ 1.38 F37A (25-37) 8.90 × 10⁴ 8.48 × 10⁻³ 95 (solution K_(D) = 172 nM) 38A (25-38A) — — No binding detected

The above data indicate that the epitope that antibody G1 binds is on the C-terminal end of human α-CGRP, and amino acids 33 and 37 on human α-CGRP are important for binding of antibody G1. Also, the amidation of residue F37 is important for binding.

Example 5: Anti-CGRP Antagonist Antibody Prevents PIH

Single-unit electrophysiological techniques were used to study the response profile of peripheral and central trigeminovascular neurons in the spinal trigeminal nucleus in response to occurrence of seizure in rats treated with fremanezumab (TEV-48125) as compared to untreated rats. Cortical electrodes were used to trace the magnitude, extent and progression of epileptiform seizures.

Surgical Preparation and Single-Unit Recording.

Single-unit recordings were obtained from neurons in the trigeminal ganglion and dorsal horn as described in previous studies (Burstein et al. (1998) J. Neurophysiol. 79: 964-82; Strassman and Levy (2006) J. Neurophysiol. 95: 1298-306; Strassman et al. (1996) Nature 384: 560-4; Zhang et al. (2010) J. Neurosci. 30: 8807-14; and Zhang et al. (2011) Ann. Neurol. 69: 855-65). Experiments were done in adult Sprague-Dawley rats (250 g to 350 g). Animals were anesthetized with urethane (1.2-1.5 g/kg), artificially ventilated with oxygen, and paralyzed. Body temperature was controlled, and end-tidal CO₂ and oxygen saturation was monitored. For ganglion recordings, four separate craniotomies were made: over the contralateral cortex, to advance the microelectrode; over the ipsilateral parietal cortex, and the ipsilateral occipital cortex, for application of the picrotoxin and recording of electrocorticogram activity; and over the ipsilateral transverse sinus, for electrical and mechanical stimulation of dural afferents, and lidocaine application. For dorsal horn recording, the same craniotomies were made over the ipsilateral cortex and the ipsilateral transverse sinus, but no contralateral craniotomy was made. Instead, a laminectomy was made to expose the upper cervical spinal cord (C1-2) for microelectrode recording. In both the trigeminal ganglion and the dorsal horn recording experiments, the search stimulus for finding dura-sensitive neurons is single-shock electrical stimulation applied to the dura covering the transverse sinus.

Seizure Induction and Electrocorticogram Recording.

Seizure was induced by application of picrotoxin to the surface of the cerebral cortex (10 μl, applied on a small piece of gelfoam, at a concentration of either 5 mM or 100 mM, for focal or generalized seizure, respectively). For verification of seizure induction, cortical activity was recorded with a glass micropipette (0.9% saline, ˜1 megohm, 7 μm tip) placed just below the surface of the cerebral cortex, at the parietal and the occipital site.

Treatment with the Monoclonal Anti-CGRP Antibody TEV-48125.

TEV-48125 (TEVA Pharmaceutical Industries Ltd., Israel) is a humanized monoclonal anti-CGRP antibody (CGRP-mAb). It was diluted in saline to a final dose of 30 mg/kg and administered intravenously (total volume 0.8 ml) four hours before induction of seizure.

Results.

In untreated animals, the induction of seizure triggered prolonged activation in peripheral and central trigeminovascular neurons. In the ganglion, activity began to increase minutes after the seizure reached their receptive fields and remained elevated for as long the seizure activity continued (FIGS. 7A-7D). In the medullary dorsal horn, 28/30 (93%) neurons were activated for over 2 hours by the seizure (FIGS. 8A-8E). In contrast, in the TEV-48125 treated animals, only 2/13 (15%) neurons were activated by the seizure (FIG. 9).

Conclusions.

This example shows activation of the trigeminovascular pathway by seizure and prevention of such activation by TEV-48125. Since the trigeminovascular pathway mediates PIH, the findings demonstrate that TEV-48125 can prevent PIH if given prophylactically. Critically, the results show that in untreated animals, the induction of seizure triggered prolonged activation in 28/30 (93%) neurons whereas in the TEV-48125 treated animals, only 2/13 (15%) neurons were activated by the seizure.

Example 6: Selective Inhibition of Trigeminovascular Neurons by the Humanized Monoclonal Anti-CGRP Antibody (Fremanezumab, TEV-48125)

The purpose of this study was to better understand how the CGRP-mAb fremanezumab (TEV-48125) modulates meningeal sensory pathways. To answer this question single-unit recording was used to determine the effects of fremanezumab (30 mg/kg IV) and a IgG2 isotype control antibody (isotype-conAb) on spontaneous and evoked activity in naïve and CSD-sensitized trigeminovascular neurons in the spinal trigeminal nucleus of anesthetized male and female rats. The study demonstrates that in both sexes fremanezumab inhibited naïve high-threshold (HT) but not wide-dynamic range trigeminovascular neurons, and that the inhibitory effects on the neurons were limited to their activation from the intracranial dura but not facial skin or cornea. Additionally, when given sufficient time, fremanezumab prevents activation and sensitization of HT neurons by cortical spreading depression.

A. Materials and Methods

Surgical Preparation

Experiments were approved by the Beth Israel Deaconess Medical Center and Harvard Medical School standing committees on animal care, and in accordance with the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male and female Sprague-Dawley rats (250-350 g) were anesthetized with urethane (0.9-1.2 g/kg i.p.). They were fitted with an intra-tracheal tube to allow artificial ventilation (0.1 L/min of O₂), and an intra-femoral-vein cannula for later infusion of drugs. Rats were placed in a stereotaxic apparatus, and core temperature was kept at 37° C. using a heating blanket. End-tidal CO₂ was continuously monitored and kept within physiological range (3.5-4.5 pCO₂). Once stabilized, rats were paralyzed with rocuronium bromide (10 mg/ml, 1 ml/hr continuous intravenous infusion) and ventilated. For stimulation of the cranial dura later in the experiment, a 5×5-mm opening was carefully carved in the parietal and occipital bones in front and behind the lambda suture, directly above the left transverse sinus. The exposed dura was kept moist using a modified synthetic interstitial fluid (135 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 5 mM CaCl₂), 10 mM glucose and 10 mM Hepes, pH 7.2). For single-unit recording in the spinal trigeminal nucleus, a segment of the spinal cord between the obex and C2 was uncovered from overlying tissues, stripped of the dura mater, and kept moist with mineral oil.

Neuronal Identification and Selection

To record neuronal activity, a tungsten microelectrode (impedance 3-4 MΩ) was lowered repeatedly into the spinal trigeminal nucleus (STN) in search of central trigeminovascular neurons receiving convergent input from the dura and facial skin. Trigeminovascular neurons were first identified based on their responses to electrical stimulation of the dura. They were selected for the study if they exhibited discrete firing bouts in response to ipsilateral electrical (0.1-3.0 mA, 0.5 msec, 0.5 Hz pulses) and mechanical (with a calibrated von Frey monofilaments) stimulation of the exposed cranial dura and to mechanical stimulation of the facial skin and cornea. Dural receptive fields were mapped by indenting the dura (with the 4.19 g VFH monofilament) at points separated by 1 mm mediolaterally and rostrocaudally. Points at which dural indentation produced a response in ≥50% of the trials were considered inside the neurons receptive field. Cutaneous receptive fields were mapped by applying innocuous and noxious mechanical stimulation to all facial skin areas and the cornea. An area was considered outside the receptive field if no stimulus produced a response in ≥50% of the trials. Responses to mechanical stimulation of the skin were determined by applying brief (10 s) innocuous and noxious stimuli to the most sensitive portion of the cutaneous receptive field. Innocuous stimuli consisted of slowly passing a soft bristled brush across the cutaneous receptive field (one 5-s brush stroke from caudal to rostral and one 5-s brush stroke from rostral to caudal) and pressure applied with a loose arterial clip. Noxious stimuli consisted of pinch with a strong arterial clip (Palecek et al., 1992, J. Neurophysiol. 67:1562-1573; Dado et al., 1994, J. Neurophysiol. 71:981-1002; Burstein et al., 1998, J. Neurophysiol. 79:964-982). More intense or prolonged stimuli were not used to avoid inducing prolonged changes in spontaneous neuronal discharge or response properties. Responses to mechanical stimulation of the cornea consisted of gentle and slow brushing strokes with a thin paintbrush (about 10 hair-follicles). Two classes of neurons were thus identified: wide-dynamic-range (WDR) neurons (incrementally responsive to brush, pressure and pinch), and high-threshold (HT) neurons (unresponsive to brush). Real-time waveform discriminator was used to create and store a template for the action potential evoked in the neuron under study by electrical pulses on the dura; spikes of activity matching the template waveform were acquired and analyzed online and offline using Spike 2 software (CED, Cambridge, UK).

Induction and Recording of Cortical Spreading Depression

Cortical spreading depression (CSD) was induced mechanically by inserting a glass micropipette (tip diameter 25 μm) about 1 mm into the visual cortex for 10 sec. At a propagation rate of 3-5 mm/min, a single wave of CSD was expected to enter the neuronal receptive field within 1-2 min of cortical stimulation. For verification of CSD, cortical activity was recorded (electrocorticogram) with a glass micropipette (0.9% saline, ˜1 megohm, 7 um tip) placed just below the surface of the cerebral cortex (approximately 100 μm). The electrocorticogram electrode was positioned about 6 mm anterior to the visual cortex.

Treatment with the Monoclonal Anti-CGRP Antibody Fremanezumab (TEV-48125)

Fremanezumab (also known as TEV-48125/LBR-101/RN-307) (TEVA Pharmaceutical Industries Ltd., Israel) is a humanized monoclonal anti-CGRP antibody (CGRP-mAb). It was diluted in saline to a final dose of 30 mg/kg and administered intravenously (bolus injection, total volume 0.6-0.7 ml). A corresponding human IgG2 isotype control antibody (isotype-conAb) was also diluted in saline to a final dose of 30 mg/kg and administered intravenously (bolus injection, total volume 1.6-2.0 ml).

Experimental Protocol

The experimental protocol included two parts. The first part was designed to compare CGRP-mAb vs isotype-conAb effects on spontaneous and induced activity of naïve trigeminovascular neurons, and the second part was designed to test CGRP-mAb vs isotype-conAb effects on the activation and sensitization of trigeminovascular neurons by CSD. Both parts included sampling of WDR and HT neurons in male and female rats. In the first part, the baseline neuronal profile was established by (a) mapping the dural, cutaneous and corneal receptive field; (b) measuring responses (mean spikes/sec) to mechanical stimulation of the dura (with a fixed force), skin (brush, pressure, pinch) and cornea (brush), and (c) measuring spontaneous firing rate (recorded over 30 min prior to treatment). Once the baseline was established, CGRP-mAb or isotype-conAb were administered and receptive fields were remapped, neuronal responses to stimulation of the dura, skin and cornea were re-examined, and the spontaneous activity rate was re-sampled at 1, 2, 3, and 4 hours post-treatment. The resulting values for each measure were then compared with the respective baseline values obtained before treatment. In the second part, CSD was induced 4 hours after administration of CGRP-mAb or isotype-conAb and 2 hours later (i.e., 6 hours after treatment) receptive field size, spontaneous activity rate, and response magnitude to stimulation of the dura, skin and cornea were measured again. The resulting post-CSD values for each measure were then compared with the respective pre-CSD values obtained at the 4-hour post-treatment time. This part was initiated only in cases in which the physiological condition of the rats (heart rate, blood pressure, respiration, end tidal CO2) and the neuronal isolation signal (signal-to-noise ratio ≥1:3) were stable at the 4-hour post-treatment time point.

At the conclusion of each experiment, a small lesion was produced at the recording site (anodal DC of 15 μA for 15 sec) and its localization in the dorsal horn was determined postmortem using histological analysis as described elsewhere (Zhang et al. (2011) Ann. Neurol. 69: 855-865). Only one neuron was studied in each animal.

Data Analysis

To calculate the response magnitude to each stimulus, the mean firing frequency occurring before the onset of the first stimulus (30 min for spontaneous activity, 10 sec for mechanical stimulation of the dura, skin and cornea) was subtracted from the mean firing frequency that occurred throughout the duration of each stimulus. In the first part of the study, corresponding values for each measure (determined at 1, 2, 3, 4 hrs after treatment) were compared with the respective baseline values obtained before fremanezumab or isotype-conAb administration. In the second part of the study, resulting values for each measure (determined 2 hours after CSD induction) were compared with the respective values obtained before CSD induction in the 2 treatment groups (fremanezumab and isotype-conAb). A neuron was considered activated when its mean firing rate after CSD exceeded its mean baseline activity by 2 standard deviations of that mean for a period >10 min, which translated to ≥33% increase in activity. A neuron was considered sensitized if 2 hours after occurrence of CSD it exhibited enhanced responses to at least 3 of the following 5 stimuli: dural indentation, brushing, pressuring or pinching the skin, and brushing the cornea. Mean firing rates of respective values were compared using nonparametric statistics (Wilcoxon signed-ranks test). Two-tailed level of significance was set at 0.05.

B. Results

The database for testing CGRP-mAb vs isotype-conAb effects on spontaneous and induced activity of naïve trigeminovascular neurons consisted of 63 neurons. Of these, 31 were classified as WDR and 32 as HT. Of the 31 WDR neurons, 18 (11 in males, 7 in females) were tested before and after administration of the CGRP-mAb, and 13 (7 in males, 6 in females) were tested before and after administration of the isotype-conAb. Of the 32 HT neurons, 18 (11 in males, 7 in female) were tested before and after administration of the CGRP-mAb, and 14 (8 in males, 6 in females) were tested before and after administration of the isotype-conAb.

The database for testing CGRP-mAb vs. isotype-conAb effects on the activation and sensitization of the neurons by CSD consisted of 50 neurons. Of these, 23 were classified as WDR and 27 as HT. Of the 23 WDR neurons, 13 (7 in males, 6 in females) were tested in the CGRP-mAb treated animals and 10 (5 in males, 5 in females) in the isotype-conAb treated animals. Of the 27 HT neurons, 14 (8 in males, 6 in female) were tested in the CGRP-mAb treated animals, and 13 (7 in males, 6 in females) in the isotype-conAb treated animals.

Recording Sites, Receptive Fields and Neuronal Classes Recording site, maps of dural and cutaneous receptive fields, and cell types did not differ between neurons tested for CGRP-mAb and those tested for the isotype-conAb (FIGS. 10A-10J). All identified recording sites were localized in laminae I-II and IV-V of the first cervical segment of the spinal cord and the caudal part of nucleus caudalis. In all cases, the most sensitive area of the dural receptive field was along the transverse sinus and the most sensitive area of the cutaneous receptive field was around the eye, involving the cornea in more than 90% of the cases.

Spontaneous Activity of Naïve Central Trigeminovascular Neurons

In male rats, intravenous administration of the CGRP-mAb reduced the spontaneous activity of the HT but not the WDR neurons (FIGS. 11A and 11B). In the HT group, neuronal firing decreased within 3-4 hrs by 90% (p=0.040). Occasionally, the firing rate of some HT neurons decreased within 1-2 hours after the intravenous administration of the CGRP-mAb (FIG. 11D). In contrast, intravenous administration of the isotype-conAb did not alter the spontaneous activity of either group of neurons (FIGS. 11E and 11F).

In females, unlike in males, intravenous administration of the CGRP-mAb did not reduce the spontaneous activity of HT or WDR neurons (FIG. 11C). Similarly, intravenous administration of the isotype-conAb did not alter the spontaneous activity of either group of neurons (FIG. 11G). Critically, the baseline (i.e., before any treatment) spontaneous firing rate of HT and WDR neurons did not differ between the male and the female rats (p=0.14). For the HT neurons, mean spikes/sec before any treatment was 1.7±1.1 in the male vs. 1.9±1.0 in the female (p=0.55). For the WDR neurons, mean spikes/sec before any treatment was 0.3±0.6 in the male vs. 2.2±1.1 in the female (p=0.16).

Sensitivity of Naïve Central Trigeminovascular Neurons to Dural Indentation

In both male and female rats, intravenous administration of the CGRP-mAb reduced the sensitivity to mechanical stimulation of the dura in the HT but not the WDR neurons (FIGS. 12A-12C). In males, the firing of HT neurons decreased by 75% (p=0.047) whereas in females it decreased by 61% (p=0.017). Regardless of the sex, intravenous administration of the isotype-conAb did not alter the sensitivity to dural stimulation in either group of neurons (FIGS. 12D-12F).

Sensitivity of Naïve Central Trigeminovascular Neurons to Mechanical Stimulation of the Periorbital Skin and the Cornea

Intravenous administration of the CGRP-mAb (FIGS. 13A-13D)—or the isotype-conAb (FIGS. 13E-13H) did not alter the responses of HT or WDR neurons to innocuous (brush, pressure) or noxious (pinch) mechanical stimulation of the skin or the cornea FIGS. 14A-14F in male or female rats.

Cortical Spreading Depression

Effects of CGRP-mAb (n=27) or isotype-conAb (n=23) on activation of central trigeminovascular neurons by CSD was tested in 50 neurons in which baseline firing rate (i.e., mean spikes/sec before induction of CSD) was reliable and consistent over hours. At baseline (i.e., before CSD), the spontaneous firing rate of HT and WDR neurons did not differ between the male and the female rats (p=0.14). For the HT neurons, mean spikes/sec before induction of CSD was 1.2±0.6 in the male vs. 3.3±1.7 in the female (p=0.29). For the WDR neurons, mean spikes/sec before induction of CSD was 1.5±0.6 in the male vs. 3.5±2.2 in the female (p=0.37).

CSD-Induced Activity in Central Trigeminovascular Neurons

In male rats, two hours after induction of CSD and 6 hours after isotype-conAb administration, the mean firing rate of the 7 HT neurons increased from 1.1±0.8 spikes/sec before CSD to 10.2±2.1 after CSD (p=0.019), whereas the mean firing rate of the 5 WDR neurons did not increase (0.5±0.3 spikes/sec before CSD vs. 1.6±0.5 after CSD; p=0.14) (FIGS. 15A and 15B). In contrast, in the CGRP-mAb treated rats, the response magnitude of the 8 HT neurons remained unchanged 2 hours after induction of CSD and 6 hours after CGRP-mAb administration (1.2±0.6 spikes/sec before CSD vs. 1.9±1.5 after CSD, p=0.29) (FIGS. 15D and 15E). In other words, the expected CSD-induced activation of the HT neurons was prevented by the CGRP-mAb treatment.

In female rats, two hours after induction of CSD and 6 hours after isotype-conAb administration, the mean firing rate of the 6 HT neurons increased from 1.9±1.0 spikes/sec before CSD to 10.0±4.5 after CSD (p=0.027), whereas the mean firing rate of the 5 WDR neurons remained unchanged (2.6±1.2 spikes/sec before CSD vs. 2.2±0.9 after CSD p=0.73) (FIG. 15C). In contrast, in the CGRP-mAb treated rats, the response magnitude of the 6 HT neurons remained unchanged 2 hours after induction of CSD and 6 hours after CGRP-mAb administration (3.3±1.7 spikes/sec before CSD vs. 5.0±3.4 after CSD, p=0.45) (FIG. 15F). As in the male, the expected CSD-induced activation of the HT neurons was prevented by the CGRP-mAb treatment.

To further examine CGRP-mAb effects on the activation of WDR and HT neurons by CSD, a case-by-case analysis was also performed. Of all CGRP-mAb and isotype-conAb treated WDR neurons, 5/13 and 4/10 were activated by CSD, a mere 2% difference. In contrast, of all CGRP-mAb and isotype-conAb treated HT neurons, 2/14 and 13/13 were activated by CSD, an 86% difference.

CSD-Induced Sensitization

Regardless of activation by CSD, 11/13 HT and none of the WDR neurons fulfilled criteria for the development of sensitization (defined in the data analysis section). Therefore, the CGRP-mAb's ability to interfere with the development of sensitization after CSD is presented for HT but not WDR neurons.

Expansion of Dural Receptive Fields and Enhanced Responses to Mechanical Stimulation of the Dura after CSD

In the isotype-conAb treated group, dural receptive fields expanded in 5/7 HT neurons in males and 6/6 HT neurons in females (FIG. 16A). Two hours after induction of CSD (6 hours after isotype-conAb administration), neuronal responses to dural indentation with VFH increased in all 7 HT neurons in the male (12.8±3.9 spikes/sec before CSD vs. 22.0±3.7 after CSD; p=0.026), and all 6 HT neurons in the female (8.5±1.7 before CSD vs. 21.6±5.1 after CSD, p=0.047) (FIGS. 17A-17C).

In contrast, in the CGRP-mAb treated group, expansion of dural receptive fields, which was smaller when it occurred, was recorded in only 2/8 HT neurons in the male and 0/6 in the female (FIG. 16B). Two hours after induction of CSD (6 hours after CGRP-mAb administration), neuronal responses to dural indentation with VFH remained unchanged in all HT neurons in both the male (1.8±0.6 before CSD vs. 1.9±1.5 after CSD, p=0.83) and the female (10.5±1.6 before CSD vs. 8.1±6.4 after CSD, p=0.72, FIGS. 17D-17F)—indicative of prevention of sensitization. Thus, the CGRP-mAb prevented the development of intracranial mechanical hypersensitivity in HT neurons in both male and female rats.

Expansion of Cutaneous Receptive Fields and Enhanced Responses to Mechanical Stimulation of the Periorbital Skin after CSD (i.e., Central Sensitization)

In the isotype-conAb treated group, facial receptive fields expanded in 5/7 HT neurons in males and 6/6 HT neurons in females (FIG. 16A). Two hours after induction of CSD (6 hours after isotype-conAb administration) responses to brush and pressure increased significantly in all 13 HT neurons (7 in males, 6 in females) (FIGS. 18A-18C). In males, responses to brush and pressure increased from 0.0 to 18.2±9.1 spikes/sec (p=0.046) and from 16.6±4.2 to 35.8±9.1 spikes/sec (p=0.045), respectively (FIG. 18B). In females, responses to brush and pressure increased from 0.0 to 8±6.5 spikes/sec (p=0.027) and from 9.3±2.7 to 31.8±13.6 spikes/sec (p=0.016), respectively (FIG. 18C). In contrast, responses to pinch increased significantly in all HT neurons in females (19.3±5.0 spikes/sec before CSD vs. 45.8±12.4 spikes/sec after CSD, n=6, p=0.027) but not in the male (33.8±7.1 spikes/sec before CSD vs. 52.4±10.3 spikes/sec after CSD, n=6, p=0.068) (FIGS. 18B and 18C).

In the CGRP-mAb treated rats, facial receptive fields expanded in only 2/8 HT neurons in males and 0/6 HT neurons in females (FIG. 16B). Two hours after induction of CSD (6 hours after CGRP-mAb administration), neuronal responses to brush (p=0.35), pressure (p=0.63) and pinch (p=0.78) remained unchanged in all HT neurons in both male and female (FIGS. 18D-18F)—suggesting that the CGRP-mAb prevented induction of sensitization.

Enhanced Responses to Corneal Stimulation after CSD

In the isotype-conAb treated rats, responses to corneal stimulation after CSD increased significantly in females (7.6±1.9 spikes/sec before CSD vs. 21.0±6.4 spikes/sec after CSD, n=6, p=0.044) but not in males (11.0±2.6 spikes/sec before CSD vs. 21.6±8.7 spikes/sec after CSD, n=7, p=0.19) HT neurons (FIGS. 19A-19C).

In the CGRP-mAb treated female rats, response to brushing the cornea remained unchanged in the 6 HT neurons (p=0.51)—suggesting prevention of sensitization; and as expected, it also remained unchanged in the 8 HT neurons in the males (10.8±3.3 spikes/sec before CSDS vs. 9.4±1.8 (spikes/sec after CSD, p=0.60) (FIGS. 19D-19F). Thus, the CGRP-mAb prevented the development of corneal hypersensitivity in HT neurons in female but not male rats.

C. Discussion

The study demonstrates that the humanized monoclonal anti-CGRP antibody fremanezumab inhibits activation and sensitization of HT but not WDR trigeminovascular neurons (FIG. 20). In males, the CGRP-mAb inhibited the spontaneous activity of naive HT neurons and their responses to stimulation of the intracranial dura but not facial skin or cornea, whereas in females it only inhibited their responses to stimulation of the intracranial dura. When given sufficient time, however, the CGRP-mAb prevented in both sexes the activation and consequential sensitization of the HT neurons by CSD, but not the partial activation of WDR neurons. Mechanistically, these findings suggest that HT neurons play a critical role (not recognized before) in the initiation of the perception of headache and the development of allodynia and central sensitization. Clinically, the present findings may help explain the therapeutic effectiveness of CGRP-mAb in preventing headaches of intracranial origin such as migraine and why this therapeutic approach may not be effective for every migraine patient.

This study tested the effects on CGRP-mAb on the responsiveness of different classes of central trigeminovascular neurons. Previously, Storer and colleagues showed that the CGRP-R antagonist BIBN4096BS inhibits naïve central trigeminovascular neurons responses to electrical stimulation of the superior sagittal sinus and microiontophoretic administration of L-glutamate (Storer et al., 2004, Br. J. Pharmacol. 142:1171-1181).

Fremanezumab Effects on HT Vs. WDR

When given intravenously, CGRP-mAb reduced baseline spontaneous activity in HT but not WDR neurons. Considering current and previous evidence that WDR trigeminovascular neurons are activated by a variety of dural stimulation used to study the pathophysiology of migraine (Davis and Dostrovsky, 1988, J. Neurophysiol. 59:648-666; Burstein et al., 1998, J. Neurophysiol. 79:964-982; Storer et al., 2004, Brit. J. Pharmacol. 142:1171-1181; Zhang et al., 2011, Ann. Neurol. 69:855-865), it is reasonable to conclude that activation of WDR alone is insufficient to induce the headache perception inepisodic migraine patients whose headaches are completely or nearly completely prevented by CGRP-mAb therapy (Bigal et al., 2015, Lancet Neurol. 14:1081-1090). Conversely, it is also reasonable to speculate that activation of WDR trigeminovascular neurons alone may be sufficient to induce the headache perception in those episodic migraine patients who do not benefit from CGRP-mAb therapy, as the headache could be unaffected by elimination of the signals sent to the thalamus from HT trigeminovascular neurons.

Outside migraine and the trigeminovascular system, HT and WDR neurons have been thought to play different roles in the processing of noxious stimuli and the perception of pain (Craig A D, 2002, Nat. Rev. Neurosci. 3:655-666; Craig A D, 2003, Trends Neurosci. 26:303-307; Craig A D, 2003, Annu. Rev. Neurosci. 26:1-30). While most HT neurons exhibit small receptive fields and respond exclusively to noxious mechanical stimuli, most WDR neurons exhibit large receptive fields and respond to both mechanical and thermal noxious stimuli (Price et al., 1976, J. Neurophysiol. 39:936-953; Price et al., 1978, J. Neurophysiol. 41:933-947; Hoffman et al., 1981, Neurophysiology 46:409-427; Dubner and Bennett, 1983, Annu. Rev. Neurosci. 6:381-418; Bushnell et al., 1984, J. Neurophysiol. 52:170-187; Surmeier et al., 1986, J. Neurophysiol. 56:328-350; Ferrington et al., 1987, J Physiol (Lond) 388:681-703; Dubner et al., 1989, J. Neurophysiol. 62:450-457; Maixner et al., 1989, J. Neurophysiol. 62:437-449; Laird and Cervero, 1991, J. Physiol. 434:561-575). Based on these differences, it is generally believed that HT neurons make a greater contribution to the spatial encoding (size, location) of pain and a lesser contribution to the encoding of pain modalities, whereas WDR neurons make a greater contribution to the radiating qualities of the pain. Along this line, it is also reasonable that those patients unresponsive to fremanezumab are the ones whose headaches affect large areas of the head (i.e., frontal, temporal, occipital, bilateral) whereas the ones whose headaches are well localized to small and distinct areas will be among the responders.

Effectiveness in Headache

Fremanezumab reduced responsiveness to mechanical stimulation of the dura (both in males and females) but not to innocuous or noxious stimulation of the skin or cornea. This finding, together with the fact that the CGRP-mAb also prevented the activation of HT trigeminovascular neurons by CSD, provides a scientific basis for fremanezumab's effectiveness in preventing headaches of intracranial origin. Conversely, lack of effects on modulating the processing of sensory and nociceptive signals that arise in the facial skin and cornea predicts that this class of drugs will have little therapeutic effect on treating prolonged trigeminal pain conditions such as dry eye and herpes-induced trigeminal neuralgia. Given that fremanezumab inhibited activation of central trigeminovascular neurons from the dura (mechanical, CSD) but not skin or cornea, and that the size of this molecule is too large to readily penetrate the blood brain barrier, it is reasonable to suggest that the inhibitory effects described above were secondary to (primary) inhibition of responses to dural indentation and CSD in peripheral trigeminovascualr neurons.

Given the wide distribution throughout the body of CGRP fibers (Kruger et al., 1988, J. Comp. Neurol. 273:149-162; Kruger et al., 1989, J. Comp. Neurol. 280:291-302; Silverman and Kruger, 1989, J. Comp. Neurol. 280:303-330), their presence in multiple spinal cord segments (Hansen et al., 2016, Pain 157:666-676; Nees et al., 2016, Pain 157:687-697), and in multiple sensory dorsal root ganglia (Edvinsson et al., 1998, J. Auton. Nerv. Syst. 70:15-22; Edvinsson et al., 2001, Microsc. Res. Techniq. 53:221-228; Cho et al., 2015, J. Korean Med. Sci. 30:1902-1910; Kestell et al., 2015, J. Comp. Neurol. 523:2555-2569; Spencer et al., 2016, J. Comp. Neurol. 524:3064-3083), it is surprising that the CGRP-mAb had little or no effect on the responses of the central neurons to noxious stimulation of the skin and cornea. If one accepts the notion that the CGRP-mAb acts mainly in the periphery, it is also reasonable to propose that peripheral aspects of the sensory innervation of the meninges and the way this innervation affects sensory transmission in the dorsal horn differ from those involved in the generation of cutaneous, corneal or other (somatic) pains. Studies on fremanezumab's effects in animal models of other pain conditions should allow for more accurate interpretation of the difference between the CGRP-mAb's effects in the dura vs. extracranial tissues not believed to have a distinct initiating role in migraine.

Inhibition of CSD-Induced Activation and Sensitization

This study demonstrates sensitization of central trigeminovascular neurons by CSD. This sensitization—observed in HT but not WDR neurons in both males and females—was prevented by the CGRP-mAb administration. These findings indicate that cutaneous allodynia in attacks preceded by aura (Burstein et al., 2000, Ann. Neurol. 47:614-624) is mediated by HT neurons that are unresponsive to innocuous mechanical stimulation of the skin at baseline (interictally in patients and before induction of CSD in animals), but become mechanically responsive to brush after the CSD. According to this scenario, among migraine aura patients, responders to the prophylactic treatment with CGRP-mAb would show no signs of cutaneous allodynia.

Male v. Female

This study also tested CGRP-mAb's effects in both male and female rats. While the overall analysis-by-sex suggests that the therapeutic benefit of this class of drugs should be similar in male and female migraineurs, it also shows that in the naïve state, CGRP-mAb reduces the spontaneous activity in male, but not female HT neurons, and that after induction of sensitization by CSD, only HT neurons recorded in females exhibited signs of sensitization to noxious stimulation of the skin and cornea. Given that migraine is more common in women than men, the differences may suggest that hyperalgesia (rather than allodynia) is more likely to develop in women than in men during migraine with aura, and that attempts to reduce neuronal excitability by CGRP-mAb in the interictal state (i.e., as a preventative), may also be more challenging in women than men. Mechanistically, the three observed differences could be attributed to greater excitability of female HT neurons, either due to these neurons' internal properties or due to differences in the strength of inputs they receive from peripheral nociceptors. Whereas no data exist to support the first option, it is possible that differences in the activation of dural immune cells and inflammatory molecules in females compared to males (McIlvried et al. (2015) Headache 55:943-957) can support the second option. Regarding fremanezumab's ability to reduce spontaneous activity in male but not female rats, one may take into consideration data showing that female rats express fewer CGRP receptors in the trigeminal ganglion and spinal trigeminal nucleus, and higher levels of CGRP-encoding mRNA in the dorsal horn (Stucky et al. (2011) Headache 51:674-692).

Finally, the inhibitory effects of CGRP-mAb required only a few hours to reach significance. This relatively short time (hours rather than days) was achieved using intravenous administration.

Example 7. Selective Inhibition of First-Order Trigeminovascular Neurons by Anti-CGRP Antibody (Fremanezumab)

A large body of evidence supports an important role for CGRP in the pathophysiology of migraine. This evidence gave rise to a global effort to develop a new generation of therapeutics that reduces the availability of CGRP in migraineurs. Recently, the second generation of such drugs, CGRP-mAb, was found to be effective in reducing the frequency of chronic or episodic migraine. In order to investigate the neural basis for this therapeutic action, the effect of fremanezumab, a CGRP-mAb, on the activity of first- and second-order neurons in the meningeal sensory pathway was tested. This study shows the effects of fremanezumab on first-order neurons in the trigeminal ganglion (FIGS. 21A-24B).

Design/Methods:

Single-unit extracellular recording techniques were used to determine the effects of fremanezumab (30 mg/kg IV) and its isotype (control) on the activity of first-order trigeminovascular neurons in the trigeminal ganglion evoked by cortical spreading depression (CSD) in urethane-anesthetized male rats. CSD was induced by pinprick 4 hours after drug/isotype infusion.

Results:

CSD induced activation of 40% of neurons tested in isotype-treated animals and 20% of neurons tested in fremanezumab-treated animals. As shown in FIG. 24A (a-delta), in isotype treated animals, CSD activated 54% of all a-delta fibers, which was similar to the percentage of a-delta fibers activated by CSD in untreated animals. In contrast in animals treated with fremanezumab, CSD activated only 14% of all a-delta fibers. This difference was statistically significant (Z test p=0.001). In isotype treated animals (FIG. 24B; C-type), CSD activated 31% of all C-fibers, which was similar to the percentage of C-fibers activated by CSD in untreated animals. Similarly, in animals treated with fremanezumab, CSD activated 23% of all C-fibers. This difference was statistically insignificant (Z test p>0.05).

Thus, the effect of fremanezumab was selective for A-delta neurons: the percentage of A-delta neurons that responded to CSD was reduced significantly (p<0.05) from 54% (isotype) to 14% (fremanezumab) (FIG. 24A), whereas the percentage of C-fibers neurons that responded to CSD showed no significant change (31% vs. 23%, isotype vs. fremanezumab) (FIG. 24B).

The selective action of fremanezumab on A-delta but not C-fiber first-order neurons can help explain the selective inhibition of second-order high-threshold but not wide-dynamic range neurons. For patients whose chronic and episodic migraines are relieved by fremanezumab, the findings raise the possibility that A-delta neurons play a critical role in the initiation and chronification of the perception of headache whereas C-fiber neurons contribute to the associated allodynia and central sensitization.

Without wishing to be bound by any particular theory, a proposed mechanism for the prevention of migraine by anti-CGRP monoclonal antibodies is provided. Briefly, CSD induces brief constriction, brief dilatation, and prolonged constriction of pial arteries, as well as immediate and delayed activation of C-fiber meningeal nociceptors containing CGRP. Upon their CGRP-independent activation, meningeal C-fibers release CGRP in the dura and by doing so, mediate a CGRP-dependent activation of the nearby Aδ-fibers. Once activated, C-fiber meningeal nociceptors converge on and activate WDR neurons in the spinal trigeminal nucleus, whereas Aδ-fibers converge on and activate both WDR and HT neurons that eventually transmit the nociceptive signals from the dura to the thalamus. The absence of CGRP receptors from the meningeal C-fibers renders the activation of the C-WDR pathway CGRP-independent, and thus, unresponsive to the anti-CGRP monoclonal antibodies. In contrast, the presence of CGRP receptors on the meningeal Aδ-fibers renders the activation of the Aδ-HT pathway CGRP-dependent, and thus, responsive to the anti-CGRP monoclonal antibodies.

Deposit of Biological Material

The following materials have been deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, USA (ATCC):

ATCC Material Antibody No. Accession No. Date of Deposit pDb.CGRP.hFcGI G1 heavy chain PTA-6867 Jul. 15, 2005 pEb.CGRP.hKGI G1 light chain PTA-6866 Jul. 15, 2005

Vector pEb.CGRP.hKGI is a polynucleotide encoding the G1 light chain variable region and the light chain kappa constant region; and vector pDb.CGRP.hFcGI is a polynucleotide encoding the G1 heavy chain variable region and the heavy chain IgG2 constant region containing the following mutations: A330P331 to S330S331 (amino acid numbering with reference to the wildtype IgG2 sequence; see Eur. J. Immunol. (1999) 29:2613-2624).

These deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Rinat Neuroscience Corp. and ATCC, which assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 USC Section 122 and the Commissioner's rules pursuant thereto (including 37 CFR Section 1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

Antibody Sequences

G1 heavy chain variable region amino acid sequence (SEQ ID NO: 1) EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWISWVRQAPGKGLEWVAEIRSESDASATHYAEAVKGRFTISRDNAKN SLYLQMNSLRAEDTAVYYCLAYFDYGLAIQNYWGQGTLVTVSS G1 light chain variable region amino acid sequence (SEQ ID NO: 2) EIVLTQSPATLSLSPGERATLSCKASKRVTTYVSWYQQKPGQAPRLLIYGASNRYLGIPARFSGSGSGTDFTLTISSLE PEDFAVYYCSQSYNYPYTFGQGTKLEIK G1 CDR H1 (extended CDR) (SEQ ID NO: 3) GFTFSNYWIS G1 CDR H2 (extended CDR) (SEQ ID NO: 4) EIRSESDASATHYAEAVKG G1 CDR H3 (SEQ ID NO: 5) YFDYGLAIQNY G1 CDR L1 (SEQ ID NO: 6) KASKRVTTYVS G1 CDR L2 (SEQ ID NO: 7) GASNRYL G1 CDR L3 (SEQ ID NO: 8) SQSYNYPYT G1 heavy chain variable region nucleotide sequence (SEQ ID NO: 9) GAAGTTCAGCTGGTTGAATCCGGTGGTGGTCTGGTTCAGCCAGGTGGTTCCCTGCGTCTGTCCTGCGCTGCTTCCGGTT TCACCTTCTCCAACTACTGGATCTCCTGGGTTCGTCAGGCTCCTGGTAAAGGTCTGGAATGGGTTGCTGAAATCCGTTC CGAATCCGACGCGTCCGCTACCCATTACGCTGAAGCTGTTAAAGGTCGTTTCACCATCTCCCGTGACAACGCTAAGAAC TCCCTGTACCTGCAGATGAACTCCCTGCGTGCTGAAGACACCGCTGTTTACTACTGCCTGGCTTACTTTGACTACGGTC TGGCTATCCAGAACTACTGGGGTCAGGGTACCCTGGTTACCGTTTCCTCC G1 light chain variable region nucleotide sequence (SEQ ID NO: 10) GAAATCGTTCTGACCCAGTCCCCGGCTACCCTGTCCCTGTCCCCAGGTGAACGTGCTACCCTGTCCTGCAAAGCTTCCA AACGGGTTACCACCTACGTTTCCTGGTACCAGCAGAAACCCGGTCAGGCTCCTCGTCTGCTGATCTACGGTGCTTCCAA CCGTTACCTCGGTATCCCAGCTCGTTTCTCCGGTTCCGGTTCCGGTACCGACTTCACCCTGACCATCTCCTCCCTGGAA CCCGAAGACTTCGCTGTTTACTACTGCAGTCAGTCCTACAACTACCCCTACACCTTCGGTCAGGGTACCAAACTGGAAA TCAAA G1 heavy chain full antibody amino acid sequence (including modified IgG2 as described herein) (SEQ ID NO: 11) EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWISWVRQAPGKGLEWVAEIRSESDASATHYAEAVKGRFTISRDNAKN SLYLQMNSLRAEDTAVYYCLAYFDYGLAIQNYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVA GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLN GKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK G1 light chain full antibody amino acid sequence (SEQ ID NO: 12) EIVLTQSPATLSLSPGERATLSCKASKRVTTYVSWYQQKPGQAPRLLIYGASNRYLGIPARFSGSGSGTDFTLTISSLE PEDFAVYYCSQSYNYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC G1 heavy chain full antibody nucleotide sequence (including modified IgG2 as described herein) (SEQ ID NO: 13) GAAGTTCAGCTGGTTGAATCCGGTGGTGGTCTGGTTCAGCCAGGTGGTTCCCTGCGTCTGTCCTGCGCTGCTTCCGGTT TCACCTTCTCCAACTACTGGATCTCCTGGGTTCGTCAGGCTCCTGGTAAAGGTCTGGAATGGGTTGCTGAAATCCGTTC CGAATCCGACGCGTCCGCTACCCATTACGCTGAAGCTGTTAAAGGTCGTTTCACCATCTCCCGTGACAACGCTAAGAAC TCCCTGTACCTGCAGATGAACTCCCTGCGTGCTGAAGACACCGCTGTTTACTACTGCCTGGCTTACTTTGACTACGGTC TGGCTATCCAGAACTACTGGGGTCAGGGTACCCTGGTTACCGTTTCCTCCGCCTCCACCAAGGGCCCATCTGTCTTCCC ACTGGCCCCATGCTCCCGCAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCAGAACCT GTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTGCAGTCCTCAGGTCTCT ACTCCCTCAGCAGCGTGGTGACCGTGCCATCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCC AAGCAACACCAAGGTCGACAAGACCGTGGAGAGAAAGTGTTGTGTGGAGTGTCCACCTTGTCCAGCCCCTCCAGTGGCC GGACCATCCGTGTTCCTGTTCCCTCCAAAGCCAAAGGACACCCTGATGATCTCCAGAACCCCAGAGGTGACCTGTGTGG TGGTGGACGTGTCCCACGAGGACCCAGAGGTGCAGTTCAACTGGTATGTGGACGGAGTGGAGGTGCACAACGCCAAGAC CAAGCCAAGAGAGGAGCAGTTCAACTCCACCTTCAGAGTGGTGAGCGTGCTGACCGTGGTGCACCAGGACTGGCTGAAC GGAAAGGAGTATAAGTGTAAGGTGTCCAACAAGGGACTGCCATCCAGCATCGAGAAGACCATCTCCAAGACCAAGGGAC AGCCAAGAGAGCCACAGGTGTATACCCTGCCCCCATCCAGAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCT GGTGAAGGGATTCTATCCATCCGACATCGCCGTGGAGTGGGAGTCCAACGGACAGCCAGAGAACAACTATAAGACCACC CCTCCAATGCTGGACTCCGACGGATCCTTCTTCCTGTATTCCAAGCTGACCGTGGACAAGTCCAGATGGCAGCAGGGAA ACGTGTTCTCTTGTTCCGTGATGCACGAGGCCCTGCACAACCACTATACCCAGAAGAGCCTGTCCCTGTCTCCAGGAAA GTAA G1 light chain full antibody nucleotide sequence (SEQ ID NO: 14) GAAATCGTTCTGACCCAGTCCCCGGCTACCCTGTCCCTGTCCCCAGGTGAACGTGCTACCCTGTCCTGCAAAGCTTCCA AACGGGTTACCACCTACGTTTCCTGGTACCAGCAGAAACCCGGTCAGGCTCCTCGTCTGCTGATCTACGGTGCTTCCAA CCGTTACCTCGGTATCCCAGCTCGTTTCTCCGGTTCCGGTTCCGGTACCGACTTCACCCTGACCATCTCCTCCCTGGAA CCCGAAGACTTCGCTGTTTACTACTGCAGTCAGTCCTACAACTACCCCTACACCTTCGGTCAGGGTACCAAACTGGAAA TCAAACGCACTGTGGCTGCACCATCTGTCTTCATCTTCCCTCCATCTGATGAGCAGTTGAAATCCGGAACTGCCTCTGT TGTGTGCCTGCTGAATAACTTCTATCCGCGCGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAAC TCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACCCTGAGCAAAGCAG ACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCTCCAGTCACAAAGAGCTTCAACCG CGGTGAGTGCTAA Amino acid sequence comparison of human and rat CGRP (human α-CGRP (SEQ ID NO: 15); human β-CGRP (SEQ ID NO: 43); rat α-CGRP (SEQ ID NO: 41); and ratβ-CGRP (SEQ ID NO: 44)): NH₂-ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF-CONH₂ (human α-CGRP) NH₂-ACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF-CONH₂ (human β-CGRP) NH₂-SCNTATCVTHRLAGLLSRSGGVVKDNFVPTNVGSEAF-CONH₂ (rat α-CGRP) NH₂-SCNTATCVTHRLAGLLSRSGGVVKDNFVPTNVGSKAF-CONH₂ (rat β-CGRP) Light chain variable region LCVR17 amino acid sequence (SEQ ID NO: 58) DIQMTQSPSSLSASVGDRVTITCRASQDIDNYLNWYQQKPGKAPKLLIYYTSEYHSGVPSRFSGSGSGTDFTFTISSLQ PEDIATYYCQQGDALPPTFGQGTKLEIK Heavy chain variable region HCVR22 amino acid sequence (SEQ ID NO: 59) QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGTGDTRYIQKFAGRVTMTRDTSTSTV YMELSSLRSEDTAVYYCARLSDYVSGFSYWGQGTLVTVSS Light chain variable region LCVR18 amino acid sequence (SEQ ID NO: 60) DIQMTQSPSSLSASVGDRVTITCRASQDIDNYLNWYQQKPGKAPKLLIYYTSEYHSGVPSRFSGSGSGTDFTFTISSLQ PEDIATYYCQQGDALPPTFGQGTKLEIK Heavy chain variable region HCVR23 amino acid sequence (SEQ ID NO: 61) QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGTGKTVYIQKFAGRVTMTRDTSTSTV YMELSSLRSEDTAVYYCARLSDYVSGFSYWGQGTLVTVSS Light chain variable region LCVR19 amino acid sequence (SEQ ID NO: 62) DIQMTQSPSSLSASVGDRVTITCRASKDISKYLNWYQQKPGKAPKLLIYYTSGYHSGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYCQQGDALPPTFGGGTKVEIK Heavy chain variable region HCVR24 amino acid sequence (SEQ ID NO: 63) QVQLVQSGAEVKKPGSSVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGTGKTVYIQKFADRVTITADKSTSTA YMELSSLRSEDTAVYYCARLSDYVSGFGYWGQGTTVTVSS Light chain variable region LCVR20 amino acid sequence (SEQ ID NO: 64) DIQMTQSPSSLSASVGDRVTITCRASRPIDKYLNWYQQKPGKAPKLLIYYTSEYHSGVPSRFSGSGSGTDFTFTISSLQ PEDIATYYCQQGDALPPTFGQGTKLEIK Heavy chain variable region HCVR25 amino acid sequence (SEQ ID NO: 65) QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGTGKTVYIQKFAGRVTMTRDTSTSTV YMELSSLRSEDTAVYYCARLSDYVSGFGYWGQGTLVTVSS Light chain variable region LCVR21 amino acid sequence (SEQ ID NO: 66) DIQMTQSPSSLSASVGDRVTITCRASQDIDKYLNWYQQKPGKAPKLLIYYTSGYHSGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYCQQGDALPPTFGGGTKVEIK Heavy chain variable region HCVR26 amino acid sequence (SEQ ID NO: 67) QVQLVQSGAEVKKPGSSVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGTGKTVYIQKFAGRVTITADKSTSTA YMELSSLRSEDTAVYYCARLSDYVSGFGYWGQGTTVTVSS Light chain variable region LCVR27 amino acid sequence (SEQ ID NO: 68) QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKVPKQLIYDASTLASGVPSRFSGSGSGTDFTLTISSL QPEDVATYYCLGSYDCTNGDCFVFGGGTKVEIKR Heavy chain variable region HCVR28 amino acid sequence (SEQ ID NO: 69) EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGINGATYYASWAKGRFTISRDNSKTTVY LQMNSLRAEDTAVYFCARGDIWGQGTLVTVSS Light chain variable region LCVR29 amino acid sequence (SEQ ID NO: 70) QVLTQSPSSLSASVGDRVTINCQASQSVYDNNYLAWYQQKPGKVPKQLIYSTSTLASGVPSRFSGSGSGTDFTLTISSL QPEDVATYYCLGSYDCSSGDCFVFGGGTKVEIKR Heavy chain variable region HCVR30 amino acid sequence (SEQ ID NO: 71) EVQLVESGGGLVQPGGSLRLSCAVSGLDLSSYYMQWVRQAPGKGLEWVGVIGINDNTYYASWAKGRFTISRDNSKTTVY LQMNSLRAEDTAVYFCARGDIWGQGTLVTVSS Light chain variable region LCVR31 amino acid sequence (SEQ ID NO: 72) QVLTQSPSSLSASVGDRVTINCQASQSVYDNNYLAWYQQKPGKVPKQLIYSTSTLASGVPSRFSGSGSGTDFTLTISSL QPEDVATYYCLGSYDCSSGDCFVFGGGTKVEIKR Heavy chain variable region HCVR32 amino acid sequence (SEQ ID NO: 73) EVQLVESGGGLVQPGGSLRLSCAVSGLDLSSYYMQWVRQAPGKGLEWVGVIGINDNTYYASWAKGRFTISRDNSKTTVY LQMNSLRAEDTAVYFCARGDIWGQGTLVTVSS Light chain variable region LCVR33 amino acid sequence (SEQ ID NO: 74) QVLTQTPSPVSAAVGSTVTINCQASQSVYHNTYLAWYQQKPGQPPKQLIYDASTLASGVPSRFSGSGSGTQFTLTISGV QCNDAAAYYCLGSYDCTNGDCFVFGGGTEVVVKR Heavy chain variable region HCVR34 amino acid sequence (SEQ ID NO: 75) QSLEESGGRLVTPGTPLTLTCSVSGIDLSGYYMNWVRQAPGKGLEWIGVIGINGATYYASWAKGRFTISKTSSTTVDLK MTSLTTEDTATYFCARGDIWGPGTLVTVSS Light chain variable region LCVR35 amino acid sequence (SEQ ID NO: 76) QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKVPKQLIYDASTLASGVPSRFSGSGSGTDFTLTISS LQPEDVATYYCLGSYDCTNGDCFVFGGGTKVEIKR Heavy chain variable region HCVR36 amino acid sequence (SEQ ID NO: 77) EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGINGATYYASWAKGRFTISRDNSKTTV YLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSS Light chain variable region LCVR37 amino acid sequence (SEQ ID NO: 78) QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSTTLGITG LQTGDEADYYCGTWDSRLSAVVFGGGTKLTVL Heavy chain variable region HCVR38 amino acid sequence (SEQ ID NO: 79) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAVISFDGSIKYSVDSVKGRFTISRDNSKNT LFLQMNSLRAEDTAVYYCARDRLNYYDSSGYYHYKYYGMAVWGQGTTVTVSS 

1. A method of preventing a post-ictal headache in a subject, the method comprising: identifying a subject who is at risk for a post-ictal headache; and administering to the subject a therapeutically effective amount of a monoclonal antibody that inhibits the calcitonin gene related peptide (CGRP) pathway, wherein the monoclonal antibody comprises a CDR H1 as set forth in SEQ ID NO:3; a CDR H2 as set forth in SEQ ID NO:4; a CDR H3 as set forth in SEQ ID NO:5; a CDR L1 as set forth in SEQ ID NO:6; a CDR L2 as set forth in SEQ ID NO:7; and a CDR L3 as set forth in SEQ ID NO:8.
 2. The method of claim 1, wherein the monoclonal antibody is administered to the subject intravenously or subcutaneously.
 3. The method of claim 1, further comprising administering one or more additional doses of the monoclonal antibody to the subject. 4-8. (canceled)
 9. The method of claim 1, wherein the administering comprises administering the antibody to the subject from a pre-filled syringe, pre-filled syringe with a needle safety device, injection pen, or auto-injector comprising a dose of the monoclonal antibody.
 10. (canceled)
 11. The method of claim 1, wherein the monoclonal antibody comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:1, and a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:2.
 12. The method of claim 1, wherein the monoclonal antibody comprises a heavy chain comprising the amino acid sequence as set forth in SEQ ID NO:11, and a light chain comprising the amino acid sequence as set forth in SEQ ID NO:12.
 13. The method of claim 1, wherein the monoclonal antibody is administered at a dose of from about 225 mg to about 900 mg.
 14. The method of claim 1, wherein the monoclonal antibody is administered at a dose of about 225 mg.
 15. The method of claim 1, wherein the monoclonal antibody is administered at a dose of about 225 mg monthly or quarterly.
 16. The method of claim 1, wherein the monoclonal antibody is administered at a dose of about 675 mg.
 17. The method of claim 16, wherein the dose is an initial dose and the method further comprises administering to the subject an additional dose of about 225 mg of the monoclonal antibody once per month in each of the two months subsequent to the month in which the initial dose is administered to the subject.
 18. The method of claim 1, wherein the monoclonal antibody is administered at a dose of about 675 mg monthly or quarterly.
 19. The method of claim 1, wherein the monoclonal antibody is administered at a dose of about 900 mg.
 20. The method of claim 1, wherein the monoclonal antibody is administered at a dose of about 900 mg monthly or quarterly.
 21. The method of claim 1, wherein the monoclonal antibody is administered in a formulation comprising the monoclonal antibody at a concentration of at least 150 mg/mL.
 22. The method of claim 1, wherein the monoclonal antibody is administered in a volume of less than 2 mL. 23-38. (canceled)
 39. The method of claim 1, wherein the monoclonal antibody is administered prior to a seizure.
 40. The method of claim 1, wherein the monoclonal antibody is administered at least about four hours prior to a seizure.
 41. The method of claim 1, wherein the subject who is at risk for a post-ictal headache has a brain infection, traumatic brain injury, personal history of a seizure, family history of epilepsy, stroke, or dementia.
 42. The method of claim 1, wherein the subject is human. 43-68. (canceled) 